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| /files/scSeq_Hefendehl/data/20230203/seu-SCT-harmony_02-02-23.rds | data/20230203/seu-SCT-harmony_02-02-23.rds |
| /files/scSeq_Hefendehl/code/custom_functions.R | code/custom_functions.R |
| /files/scSeq_Hefendehl/data/Copy of DEG_control_vs_stroke_vs_distal_original.xlsx | data/Copy of DEG_control_vs_stroke_vs_distal_original.xlsx |
| /files/scSeq_Hefendehl/output/cluster_desiree_orginal.pdf | output/cluster_desiree_orginal.pdf |
| /files/scSeq_Hefendehl/output/Ellbowplot.svg | output/Ellbowplot.svg |
| /files/scSeq_Hefendehl/output/Gene_list_based_Clustertree.svg | output/Gene_list_based_Clustertree.svg |
| /files/scSeq_Hefendehl/output/Gene_list_based_Clusters.svg | output/Gene_list_based_Clusters.svg |
| /files/scSeq_Hefendehl/output/Gene_list_based_Clusters_byCondition.svg | output/Gene_list_based_Clusters_byCondition.svg |
| /files/scSeq_Hefendehl/output/Gene_list_based_Clusters_byHomeostatgenes.svg | output/Gene_list_based_Clusters_byHomeostatgenes.svg |
| /files/scSeq_Hefendehl/output/All_genes_Clustertree.svg | output/All_genes_Clustertree.svg |
| /files/scSeq_Hefendehl/output/All_genes_Clusters.svg | output/All_genes_Clusters.svg |
| /files/scSeq_Hefendehl/output/All_genes_Clusters_byCondition.svg | output/All_genes_Clusters_byCondition.svg |
| /files/scSeq_Hefendehl/output/All_genes_Clusters_byHomeostatgenes.svg | output/All_genes_Clusters_byHomeostatgenes.svg |
| /files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters.svg | output/Opt_All_genes_all_cells_Clusters.svg |
| /files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters_byPlate.svg | output/Opt_All_genes_all_cells_Clusters_byPlate.svg |
| /files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters_byCondition.svg | output/Opt_All_genes_all_cells_Clusters_byCondition.svg |
| /files/scSeq_Hefendehl/output/G2M_Opt_All_genes_all_cells_Clusters.svg | output/G2M_Opt_All_genes_all_cells_Clusters.svg |
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| /files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters.svg | output/Celltypes_Opt_All_genes_all_cells_Clusters.svg |
| /files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters_byPlate.svg | output/Celltypes_Opt_All_genes_all_cells_Clusters_byPlate.svg |
| /files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters_byCondition.svg | output/Celltypes_Opt_All_genes_all_cells_Clusters_byCondition.svg |
| /files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters.svg | output/Microglia_Opt_All_genes_Clusters.svg |
| /files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters_byPlate.svg | output/Microglia_Opt_All_genes_Clusters_byPlate.svg |
| /files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters_byCondition.svg | output/Microglia_Opt_All_genes_Clusters_byCondition.svg |
| /files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Condition.svg | output/Microglia_Opt_All_genes_Condition.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM1_calling.svg | output/Microglia_DAM1_calling.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM1_bygenotype.svg | output/Microglia_DAM1_bygenotype.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM1_thresholding.svg | output/Microglia_DAM1_thresholding.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM1_scoring.svg | output/Microglia_DAM1_scoring.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM1_binary.svg | output/Microglia_DAM1_binary.svg |
| /files/scSeq_Hefendehl/output/Microglia_DAM2_calling.svg | output/Microglia_DAM2_calling.svg |
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| /files/scSeq_Hefendehl/output/Microglia_HomeostatGenes_Featureplot.svg | output/Microglia_HomeostatGenes_Featureplot.svg |
| /files/scSeq_Hefendehl/output/Microglia_Homeo_calling.svg | output/Microglia_Homeo_calling.svg |
| /files/scSeq_Hefendehl/output/Microglia_Homeo_bygenotype.svg | output/Microglia_Homeo_bygenotype.svg |
| /files/scSeq_Hefendehl/output/Microglia_Homeo_thresholding.svg | output/Microglia_Homeo_thresholding.svg |
| /files/scSeq_Hefendehl/output/Microglia_Homeo_scoring.svg | output/Microglia_Homeo_scoring.svg |
| /files/scSeq_Hefendehl/output/Microglia_Homeo_binary.svg | output/Microglia_Homeo_binary.svg |
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| /files/scSeq_Hefendehl/docs/Pseudotime_linages.svg | docs/Pseudotime_linages.svg |
| /files/scSeq_Hefendehl/docs/Pseudotime_linages.pdf | docs/Pseudotime_linages.pdf |
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| /files/scSeq_Hefendehl/docs/Pseudotime1_Top10up_down_reg_Genes-correct.svg | docs/Pseudotime1_Top10up_down_reg_Genes-correct.svg |
| /files/scSeq_Hefendehl/docs/Pseudotime2.svg | docs/Pseudotime2.svg |
| /files/scSeq_Hefendehl/docs/Pseudotime2_Top10up_down_reg_Genes.svg | docs/Pseudotime2_Top10up_down_reg_Genes.svg |
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| /files/scSeq_Hefendehl/output/net.rds | output/net.rds |
| /files/scSeq_Hefendehl/output/powertable.rds | output/powertable.rds |
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| /files/scSeq_Hefendehl/output/tmp.RData | output/tmp.RData |
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if you want to save a plot use ggsave(filename = “./output/PNametheplot.svg”, plotobject))
DAM_up <- c("Itgax","Cst7","Ccl4","Clec7a","Lpl","Siglec1","Spp1","Axl","Apoe","Trem2","Csf1","Lyz2","H2-D1","Tyrobp","Cd74","Gpnmb","B2m","Cd9","Ctsb","Ctsd") # genes up regulated in DAMs versus homestatics
# additions
DAM1_up <- c("Tyrobp", "Ctsb", "Ctsd", "Apoe", "B2m", "Fth1", "Lyz2") # genes up regulated in DAM1 vs Homeostasis
DAM1_down <- c("Cx3cr1", "P2ry12", "Tmem119")# genes down regulated in DAM1 vs homeostasis
DAM2_up <- c("Trem2", "Axl", "Cst7", "Ctsl", "Lpl", "Cd9", "Csf1", "Ccl6", "Itgax", "Clec7a", "Lilrb4", "Timp2")
DAM1vsDAM2 = c("Trem2", "Cst7", "Spp1", "Lpl", "Itgax", "Ctsl", "Ctsz", "Cd68", "Axl", "Cd9", "Ccl6", "Csf1")
homeostat.genes=c("P2ry12","P2ry13","Tmem119","Cx3cr1","Selplg","Cd33")
cluster.genes=unique(c(DAM_up, DAM1vsDAM2, homeostat.genes))
ngenes = 50
distal_dat = openxlsx::read.xlsx("/files/scSeq_Hefendehl/data/Copy of DEG_control_vs_stroke_vs_distal_original.xlsx", sheet = 1)
distal_dat <- distal_dat[order(distal_dat$score, decreasing = T)[1:ngenes],]
distal_genes <- distal_dat$X1
control_dat = openxlsx::read.xlsx("/files/scSeq_Hefendehl/data/Copy of DEG_control_vs_stroke_vs_distal_original.xlsx",sheet = 2)
control_dat <- control_dat[order(control_dat$score, decreasing = T)[1:ngenes],]
control_genes <- control_dat$X1
Stroke_dat = openxlsx::read.xlsx("/files/scSeq_Hefendehl/data/Copy of DEG_control_vs_stroke_vs_distal_original.xlsx",sheet = 3)
Stroke_dat <- Stroke_dat[order(Stroke_dat$score, decreasing = T)[1:ngenes],]
Stroke_genes <- Stroke_dat$X1
Some cells appear to have a low expression of Aif1 as can be seen in the purple shading. However, some of these cells with the low expression seem to be missing later on (see Clustering example). Please check if really only cells with an expression of 0 Aif1 have been excluded
# cluster all cells for visualization purpose prior to exclusion
DefaultAssay(samples.integrated) = "RNA"
samples.integrated <- ScaleData(samples.integrated)
Centering and scaling data matrix
samples.integrated <- FindVariableFeatures(samples.integrated)
samples.integrated<- RunHarmony(samples.integrated, group.by.vars="Plate", npcs = 50)
Warning: Warning: The parameters do_pca and npcs are deprecated. They will be ignored for this function call and please remove parameters do_pca and npcs and pass to harmony cell_embeddings directly.
This warning is displayed once per session.
Transposing data matrix
Initializing state using k-means centroids initialization
Harmony 1/10
Harmony 2/10
Harmony 3/10
Harmony 4/10
Harmony 5/10
Harmony 6/10
Harmony 7/10
Harmony 8/10
Harmony converged after 8 iterations
samples.integrated<- RunPCA(samples.integrated, npcs = 50)
PC_ 1
Positive: S100a8, S100a9, S100a11, Slfn1, Cxcr2, Ifitm2, Fxyd5, Clec4d, Mmp9, Mcemp1
Cd44, Ifitm1, Gsr, Iqgap1, Sell, Hp, Slpi, Pglyrp1, Cd24a, Il1r2
Gda, Gsn, Lrg1, Mxd1, Emb, Adgre5, 4833407H14Rik, Anxa2, Sirpb1c, Lilr4b
Negative: Gm10800, Gm26870, Gm10801, Tmem206, Cd83, Xist, Apoe, P3h2, Prmt9, Cdc23
Golt1b, Zfp62, Cst7, Entpd6, Ptprm, Zfp280d, Gna11, Gm42944, Gnpda2, Lpl
Dnajc18, Lman2l, Rnf139, Zfp746, Sec22b, Ufl1, Smg5, Gm28265, Nrep, Slc33a1
PC_ 2
Positive: Cybb, Clec12a, Mki67, Cdca3, Ly6c2, Napsa, Chil3, Ifitm6, Mgst1, S100a4
Serpinb1a, Ccna2, Ccnb2, Crip1, Ube2c, Lcn2, Cenpa, Stmn1, Arhgef39, Top2a
H2-Eb1, Nusap1, Aurka, H2-Ab1, Smc2, Wfdc21, Lgals1, Plac8, H2-Aa, Cd74
Negative: Clec4d, Cxcr2, 4833407H14Rik, Il1f9, Il1b, Ifitm1, Sirpb1b, Sfxn5, Slfn1, H2-Q10
Cd300ld, Gm26809, Slfn4, Mirt1, Acod1, Amer2, Mxd1, Mmp9, Tnfaip2, Il1rn
Il18rap, Ccr1, Sirpb1c, Sirpb1a, Osgin1, Pla2g7, Il1r2, Sell, Gm5150, Hcar2
PC_ 3
Positive: Wfdc21, Lcn2, Serpinb1a, Cdca3, Hp, Ly6c2, S100a9, Chil3, Mki67, Ccnb2
Top2a, Pglyrp1, Ube2c, Ccna2, Stmn1, Hsd11b1, Ngp, S100a8, Mgst1, Arhgef39
Ltf, Ltb4r1, Smc2, Anxa1, Knl1, Chil1, Spc25, Gm10801, Nusap1, Cdca8
Negative: Cd74, H2-Eb1, Crip1, H2-Aa, H2-Ab1, S100a4, Apoe, Ccr2, Ciita, Mndal
Lgals1, Ahnak, Ifi211, Ifi47, Emilin2, Olfm1, Wfdc17, Pltp, Fxyd5, Lyz2
Cst7, Ifi209, Slamf7, Apoc2, Ifit3, Acot7, Usp18, Itgb7, Lgals3, Oas2
PC_ 4
Positive: Gm10801, Gm26870, Gm10800, S100a4, Ccr2, Ahnak, Itgb7, Ciita, Gm42944, Slc9a7
Gimap3, Cd3e, AC138284.4, Emb, Adamts6, Gimap5, Mndal, Lat, Gm28265, Zfp729a
Zfp280d, Tecpr2, 1110002L01Rik, Tbc1d10c, Gm2916, Ccdc88c, Prmt9, Tgfbi, C130089K02Rik, Rasgrp1
Negative: Apoe, Cst7, Lyz2, Ccl6, Ccl3, Ch25h, Cd83, Lilrb4a, Spp1, Ccl4
Actr3b, Hcar2, Ccnb2, Cybb, Apoc1, Wfdc17, Lpl, Cdkn1a, Egr2, Arrdc4
Ccna2, Aurka, Wfdc21, Dcstamp, Ube2c, C3, Gpx3, Cd74, Stmn1, Mki67
PC_ 5
Positive: Ccnb2, Ube2c, Kif22, Smc2, Stmn1, Srgn, Cdk1, Nusap1, Aurka, Ccna2
Mki67, Top2a, Spc25, Bub1, Cep55, Ufl1, Zfp62, Cdca3, Mcm5, Hmgn2
Jak1, Haspin, Cdkn1a, Smc4, Gm38287, Zfp41, Cks1b, Knl1, Zfp758, Kif20a
Negative: Gm10801, Gm10800, Gm26870, Gm28265, Ltf, Ifitm6, Ly6c2, Ngp, Serpinb1a, Mgst1
Lcn2, Chil3, Itgb2l, Wfdc21, Fpr2, Camp, I830127L07Rik, Clec12a, Cd177, Hp
Plac8, Nanos1, AC147565.1, Pilrb2, Ntm, Serpinb10, Ltb4r1, Rab44, Pglyrp1, Napsa
ElbowPlot(samples.integrated, ndims = 50)

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
samples.integrated <- JackStraw(samples.integrated, assay = "RNA")
samples.integrated <- ScoreJackStraw(samples.integrated, dims = 1:20)
JackStrawPlot(samples.integrated, dims = 1:20)
Warning: Removed 31636 rows containing missing values (`geom_point()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
samples.integrated<- RunUMAP(samples.integrated, reduction = "harmony", dims = 1:20)
Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
08:20:13 UMAP embedding parameters a = 0.9922 b = 1.112
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:20:13 Read 905 rows and found 20 numeric columns
08:20:13 Using Annoy for neighbor search, n_neighbors = 30
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:20:13 Building Annoy index with metric = cosine, n_trees = 50
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
08:20:13 Writing NN index file to temp file /tmp/RtmpajYTwQ/file15543b3bb31
08:20:13 Searching Annoy index using 1 thread, search_k = 3000
08:20:14 Annoy recall = 100%
08:20:15 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
08:20:17 Initializing from normalized Laplacian + noise (using RSpectra)
08:20:17 Commencing optimization for 500 epochs, with 35796 positive edges
08:20:19 Optimization finished
a = DimPlot(samples.integrated, reduction = "umap")
b = FeaturePlot(samples.integrated, reduction = "umap", features = "Aif1")
b2 = FeaturePlot(samples.integrated, reduction = "umap", features = "Ptprc")
c = RidgePlot(samples.integrated, features = "Aif1")
c2 = RidgePlot(samples.integrated, features = "Ptprc")
d = DimPlot(samples.integrated, reduction = "umap", split.by = "Genotype_Treatment", pt.size = 1)
e = FeaturePlot(samples.integrated, reduction = "umap", features = "Aif1", split.by = "Genotype_Treatment")
e2 = FeaturePlot(samples.integrated, reduction = "umap", features = "Ptprc", split.by = "Genotype_Treatment")
a|b|b2

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c|c2
Picking joint bandwidth of 0.194
Picking joint bandwidth of 0.352

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
getwd() # checks where you are run it directly in the console not here
[1] "/files/scSeq_Hefendehl"
ggsave("/files/scSeq_Hefendehl/output/cluster_desiree_orginal.pdf", a)
Saving 8 x 4 in image
d

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
e

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
e2

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
samples.integrated.filt<- samples.integrated
# no filtering of Aif1 or Ptpcr uncomment section if you want to filter
#samples.integrated.filt <- subset(x = samples.integrated.filt, subset = Aif1 > 0)
#samples.integrated.filt <- subset(x = samples.integrated.filt, subset = Ptprc > 0)
sex.genes <- rownames(samples.integrated.filt) %in% annotations$gene_name[grepl("X|Y", annotations$seq_name)]
Gmgenes <- grepl("^Gm", rownames(samples.integrated.filt))
Rikgenes <- grepl(".*Rik$", rownames(samples.integrated.filt))
samples.integrated.filt<- samples.integrated.filt[!(sex.genes|Gmgenes|Rikgenes),]
DefaultAssay(samples.integrated.filt)<- "RNA"
ifnb.list <- SplitObject(samples.integrated, split.by = "Plate")
ifnb.list <- lapply(X = ifnb.list, FUN = function(x) {
x <- NormalizeData(x)
x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 2000)
})
features <- SelectIntegrationFeatures(object.list = ifnb.list)
feat.anchors <- FindIntegrationAnchors(object.list = ifnb.list, anchor.features = features)
Scaling features for provided objects
Finding all pairwise anchors
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 814 anchors
Filtering anchors
Warning in FilterAnchors(object = object.pair, assay = assay, slot = slot, :
Number of anchor cells is less than k.filter. Retaining all anchors.
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 941 anchors
Filtering anchors
Retained 895 anchors
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 841 anchors
Filtering anchors
Warning in FilterAnchors(object = object.pair, assay = assay, slot = slot, :
Number of anchor cells is less than k.filter. Retaining all anchors.
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 927 anchors
Filtering anchors
Retained 849 anchors
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 832 anchors
Filtering anchors
Warning in FilterAnchors(object = object.pair, assay = assay, slot = slot, :
Number of anchor cells is less than k.filter. Retaining all anchors.
Running CCA
Merging objects
Finding neighborhoods
Finding anchors
Found 958 anchors
Filtering anchors
Retained 869 anchors
samples.integrated <- IntegrateData(anchorset = feat.anchors)
Merging dataset 2 into 3
Extracting anchors for merged samples
Finding integration vectors
Finding integration vector weights
Integrating data
Merging dataset 1 into 3 2
Extracting anchors for merged samples
Finding integration vectors
Finding integration vector weights
Integrating data
Merging dataset 4 into 3 2 1
Extracting anchors for merged samples
Finding integration vectors
Finding integration vector weights
Integrating data
samples.integrated.filt <- SCTransform(samples.integrated.filt, ncells = 3000)
Running SCTransform on assay: RNA
vst.flavor='v2' set. Using model with fixed slope and excluding poisson genes.
Calculating cell attributes from input UMI matrix: log_umi
Variance stabilizing transformation of count matrix of size 17541 by 905
Model formula is y ~ log_umi
Get Negative Binomial regression parameters per gene
Using 2000 genes, 905 cells
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Second step: Get residuals using fitted parameters for 17541 genes
Computing corrected count matrix for 17541 genes
Calculating gene attributes
Wall clock passed: Time difference of 8.391823 secs
Determine variable features
Centering data matrix
Place corrected count matrix in counts slot
Warning: Different cells and/or features from existing assay SCT
Set default assay to SCT
samples.integrated.filt <- ScaleData(object = samples.integrated.filt)
Centering and scaling data matrix
samples.integrated.filt <- FindVariableFeatures(samples.integrated.filt)
samples.integrated.filt <- RunPCA(samples.integrated.filt, npcs = 50)
PC_ 1
Positive: Vmn1r13, Olfr907, Rfx7, Pde6a, Pecam1, AC164437.4, Slc6a20b, Eva1c, Cux2, Slc39a2
Sass6, AC121793.2, Lipe, CT009480.1, Klkb1, AC162938.1, Cep97, Mcc, Tnfrsf14, AC153506.3
AC125532.1, AC121793.1, Galnt17, Srd5a3, Dock10, Gabra2, Pi4kb, AC127302.1, Rbm4b, Eif2a
Negative: Ctsd, Fth1, Ftl1, Lyz2, AY036118, Apoe, Lgals3bp, mt-Co3, mt-Atp6, Lag3
Gapdh, Cst7, mt-Nd1, P2ry13, Cd14, Tmem176b, mt-Rnr2, Csf3r, Rack1, Cd83
Ccl6, Actg1, Clec7a, Ank, Mdh2, Ighm, mt-Co2, Tcap, Axl, Tnfrsf12a
PC_ 2
Positive: S100a8, Ifitm2, S100a11, S100a9, Anxa2, S100a6, Ftl1, Mcemp1, Gsr, Slfn1
Gsn, Il1r2, Lilrb4a, Mmp9, Ifitm1, Cd44, Clec4d, Fxyd5, Lsp1, Anxa1
Adgre5, Lilr4b, Wfdc17, Cdk2ap2, Hp, Zyx, Plbd1, Fth1, Fgr, Lgals3
Negative: Olfr1307, AC122850.1, CT010444.1, Olfr635, AC153522.1, AC164629.1, AC127302.1, Olfr46, D10Wsu102e, AC132221.1
AC158633.2, AC125532.1, Kcnq1ot1, AC159625.1, AC162934.3, AC153887.2, AC153532.1, Olfr1336, AC161254.1, AC152979.2
CT025624.1, AC164629.5, AC157654.1, AC102369.2, AC160336.1, Hsd3b2, AC154517.2, AC153950.1, AC164629.4, AC174797.1
PC_ 3
Positive: S100a8, S100a9, S100a11, Cd24a, Slpi, Lrg1, Mmp9, Cxcr2, S100a6, Mcemp1
Hp, Ifitm1, Slfn1, Clec4d, Retnlg, Sell, Iqgap1, Pglyrp1, Il1r2, Adgre5
Cd44, Fxyd5, Gda, Gsr, Ifitm2, Sirpb1b, Slfn4, Clec4e, Wfdc21, Sirpb1c
Negative: Bhlhe41, P2ry13, Ctsd, Tanc2, Lag3, Mrpl23-ps1, Tspan14, Tmem176b, Cd14, Tmem206
AC149090.1, Cryl1, Foxn3, Lgals3bp, Mgat4a, Cd83, Gnaz, Gpr84, mt-Nd1, St6galnac4
Etv5, Pik3r1, Phgdh, Trmt2a, mt-Co3, Nop56, Kcnk13, Pcna, Rrp7a, Slc46a1
PC_ 4
Positive: Cst7, Apoe, Cd74, Axl, Lgals3bp, Clec7a, Lpl, Lyz2, mt-Nd1, Ank
Capg, H2-Ab1, Gapdh, mt-Co3, Ftl1, Itgax, Ccl3, AY036118, Ifitm3, H2-Aa
Ccl6, Rack1, S100a4, mt-Nd5, Ch25h, Aplp2, Myo5a, H2-Eb1, Tpi1, Lgals1
Negative: AC153506.3, Gnaz, CT009480.1, Olfr907, Cep97, Slc6a20b, Pecam1, Mrpl23-ps1, AC162938.1, Tug1
Slc39a2, Dock10, Sass6, Bmt2, Klkb1, Pde6a, Eva1c, Rbm4b, Tulp1, Lipe
Bhlhe41, Tnfrsf14, Galnt17, Kdsr, AC166060.1, Cux2, AC164437.4, Mrpl19, AC161763.1, Eif2a
PC_ 5
Positive: P2ry13, Tanc2, Actg1, AC124562.2, Mgat4a, Nfam1, AC149090.1, AC160130.1, Pik3r1, Tspan14
AC152414.1, Gnaz, Snx29, AC161250.2, Zfp638, BC035044, Csf3r, Etv5, Zfp292, Ccnd2
Zcchc7, Mtmr6, Kif21b, Mrpl23-ps1, Hjurp, Nfkbia, AC165106.3, Tgm2, Tlr6, Iffo1
Negative: AC121793.2, Mcc, Tnfrsf14, Apoe, Pi4kb, Slc39a2, AC161763.1, AC164437.4, Klkb1, Lipe
Cux2, Cst7, Eva1c, Tcf7l2, Sass6, Mrpl19, Pecam1, Cbx1, Atp1a2, Pde6a
Eif2a, AC162938.1, Clec7a, Axl, Rfx7, Kdsr, Asl, Rbm4b, Slc6a20b, Galnt17
samples.integrated.filt <- RunHarmony(samples.integrated.filt, group.by.vars="Plate")
Transposing data matrix
Initializing state using k-means centroids initialization
Harmony 1/10
Harmony 2/10
Harmony 3/10
Harmony 4/10
Harmony 5/10
Harmony 6/10
Harmony 7/10
Harmony 8/10
Harmony 9/10
Harmony 10/10
p <- ElbowPlot(samples.integrated.filt, ndims = 50, reduction = "harmony")
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Ellbowplot.svg", p)
Saving 4 x 4 in image
large difference in cell distribution between the genotypes
reslist= seq(0.2,1.2,0.2)
DefaultAssay(samples.integrated.filt)<- "SCT"
samples.integrated.filt.target <- RunUMAP(samples.integrated.filt, reduction = "pca", features = cluster.genes) %>%
FindNeighbors(features=cluster.genes) %>% FindClusters(features=cluster.genes, resolution = reslist)
08:21:41 UMAP embedding parameters a = 0.9922 b = 1.112
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:21:41 Read 905 rows and found 30 numeric columns
08:21:41 Using Annoy for neighbor search, n_neighbors = 30
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:21:41 Building Annoy index with metric = cosine, n_trees = 50
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
08:21:41 Writing NN index file to temp file /tmp/RtmpajYTwQ/file15561b4e5fd
08:21:41 Searching Annoy index using 1 thread, search_k = 3000
08:21:41 Annoy recall = 100%
08:21:42 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
08:21:44 Initializing from normalized Laplacian + noise (using RSpectra)
08:21:44 Commencing optimization for 500 epochs, with 41882 positive edges
08:21:46 Optimization finished
Computing nearest neighbor graph
Computing SNN
Warning: The following arguments are not used: features
Warning: The following arguments are not used: features
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8927
Number of communities: 6
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8497
Number of communities: 8
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8179
Number of communities: 9
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7891
Number of communities: 9
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7607
Number of communities: 10
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7372
Number of communities: 10
Elapsed time: 0 seconds
library(clustree)
clustreeplot <- clustree(samples.integrated.filt.target, prefix = "SCT_snn_res.")
clustreeplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
Idents(samples.integrated.filt.target) = "SCT_snn_res.0.6"
a<- DimPlot(samples.integrated.filt.target)
b <- DimPlot(samples.integrated.filt.target, split.by = "Genotype_Treatment")
c<- FeaturePlot(samples.integrated.filt.target,features = homeostat.genes)
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Gene_list_based_Clustertree.svg", clustreeplot)
Saving 8 x 8 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Gene_list_based_Clusters.svg", a)
Saving 8 x 8 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Gene_list_based_Clusters_byCondition.svg", b)
Saving 8 x 8 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Gene_list_based_Clusters_byHomeostatgenes.svg", c)
Saving 8 x 8 in image
reslist= seq(0.2,1.2,0.2)
DefaultAssay(samples.integrated.filt)<- "SCT"
samples.integrated.filt <- RunUMAP(samples.integrated.filt, reduction = "pca", dims=1:15) %>%
FindNeighbors() %>% FindClusters(resolution = reslist)
08:21:55 UMAP embedding parameters a = 0.9922 b = 1.112
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:21:55 Read 905 rows and found 15 numeric columns
08:21:55 Using Annoy for neighbor search, n_neighbors = 30
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:21:55 Building Annoy index with metric = cosine, n_trees = 50
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
08:21:55 Writing NN index file to temp file /tmp/RtmpajYTwQ/file1552b268a33
08:21:55 Searching Annoy index using 1 thread, search_k = 3000
08:21:55 Annoy recall = 100%
08:21:56 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
08:21:58 Initializing from normalized Laplacian + noise (using RSpectra)
08:21:58 Commencing optimization for 500 epochs, with 35140 positive edges
08:22:00 Optimization finished
Computing nearest neighbor graph
Computing SNN
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8927
Number of communities: 6
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8497
Number of communities: 8
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8179
Number of communities: 9
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7891
Number of communities: 9
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7607
Number of communities: 10
Elapsed time: 0 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.7372
Number of communities: 10
Elapsed time: 0 seconds
clustreeplot<-clustree(samples.integrated.filt, prefix = "SCT_snn_res.")
Idents(samples.integrated.filt) = "SCT_snn_res.0.6"
a<-DimPlot(samples.integrated.filt)
b<-DimPlot(samples.integrated.filt, split.by = "Genotype_Treatment")
c<-FeaturePlot(samples.integrated.filt,features = homeostat.genes)
clustreeplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/All_genes_Clustertree.svg", clustreeplot)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/All_genes_Clusters.svg", a)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/All_genes_Clusters_byCondition.svg", b)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/All_genes_Clusters_byHomeostatgenes.svg", c)
Saving 7 x 5 in image
samples.integrated.filt <- RunUMAP(samples.integrated.filt, reduction = "pca", dims=1:15) %>%
FindNeighbors() %>% FindClusters(resolution = 0.6)
08:22:08 UMAP embedding parameters a = 0.9922 b = 1.112
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:22:08 Read 905 rows and found 15 numeric columns
08:22:08 Using Annoy for neighbor search, n_neighbors = 30
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:22:08 Building Annoy index with metric = cosine, n_trees = 50
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
08:22:08 Writing NN index file to temp file /tmp/RtmpajYTwQ/file1557ca23415
08:22:08 Searching Annoy index using 1 thread, search_k = 3000
08:22:08 Annoy recall = 100%
08:22:09 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
08:22:11 Initializing from normalized Laplacian + noise (using RSpectra)
08:22:11 Commencing optimization for 500 epochs, with 35140 positive edges
08:22:13 Optimization finished
Computing nearest neighbor graph
Computing SNN
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 905
Number of edges: 27460
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8179
Number of communities: 9
Elapsed time: 0 seconds
a<-DimPlot(samples.integrated.filt, group.by = "seurat_clusters", pt.size =1, label = T) + NoLegend()
b<-DimPlot(samples.integrated.filt, reduction = "umap", split.by = "Plate", pt.size = 1)
c<-DimPlot(samples.integrated.filt, reduction = "umap", split.by = "Genotype_Treatment", pt.size =1)
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters.svg", a)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters_byPlate.svg", b)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Opt_All_genes_all_cells_Clusters_byCondition.svg", c)
Saving 7 x 5 in image
cell_cycle_markers <- dplyr::left_join(cell_cycle_genes, annotations, by = c("geneID" = "gene_id"))
# Acquire the S phase genes
s_genes <- cell_cycle_markers %>%
dplyr::filter(phase == "S") %>%
pull("gene_name")
# Acquire the G2M phase genes
g2m_genes <- cell_cycle_markers %>%
dplyr::filter(phase == "G2/M") %>%
pull("gene_name")
samples.integrated.filt<-CellCycleScoring(samples.integrated.filt, s.features = s_genes, g2m.features = g2m_genes)
Warning: The following features are not present in the object: Pola1, Gmnn, not
searching for symbol synonyms
Warning: The following features are not present in the object: Gtse1, not
searching for symbol synonyms
a<-DimPlot(samples.integrated.filt, reduction = "umap", group.by = "Phase", pt.size =1)
b<-DimPlot(samples.integrated.filt, reduction = "umap", group.by = "Phase", split.by = "Plate", pt.size =1)
c<-DimPlot(samples.integrated.filt, reduction = "umap", group.by = "Phase", split.by = "Genotype_Treatment", pt.size =1)
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/G2M_Opt_All_genes_all_cells_Clusters.svg", a)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/G2M_Opt_All_genes_all_cells_Clusters_byPlate.svg", b)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/G2M_Opt_All_genes_all_cells_Clusters_byCondition.svg", c)
Saving 7 x 5 in image
ref.sce.mm <- celldex::MouseRNAseqData()
snapshotDate(): 2022-10-31
see ?celldex and browseVignettes('celldex') for documentation
loading from cache
see ?celldex and browseVignettes('celldex') for documentation
loading from cache
sce.filt<- as.SingleCellExperiment(samples.integrated.filt, assay = "SCT")
pred.mouseRNA <- SingleR(test =sce.filt, ref = ref.sce.mm, assay.type.test=1,
labels = ref.sce.mm$label.fine)
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
table(pred.mouseRNA$labels) #fine tuned cell types
aNSCs Ependymal Granulocytes Macrophages
1 1 25 6
Microglia Microglia activated Monocytes Neurons activated
852 5 10 1
NK cells T cells
1 3
#table(pred.mouseRNA$pruned.labels) #cell type after pruning
samples.integrated.filt$Immgen_sc_labels_agc <- NA
samples.integrated.filt$Immgen_sc_labels_agc <- pred.mouseRNA$labels
plotScoreHeatmap(pred.mouseRNA, show.labels = TRUE,
annotation_col=data.frame(cluster=samples.integrated.filt$seurat_clusters,
row.names=rownames(pred.mouseRNA)))
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
Idents(samples.integrated.filt)<- "seurat_clusters"
a<- DimPlot(samples.integrated.filt, reduction="umap", group.by="Immgen_sc_labels_agc",
label = F)
b<- DimPlot(samples.integrated.filt, reduction="umap", group.by="Immgen_sc_labels_agc",
label = F, split.by = "Plate")
c<- DimPlot(samples.integrated.filt, reduction="umap", group.by="Immgen_sc_labels_agc", split.by = "Genotype_Treatment",
label = F)
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters.svg", a)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters_byPlate.svg", b)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Celltypes_Opt_All_genes_all_cells_Clusters_byCondition.svg", c)
Saving 7 x 5 in image
samples.integrated.filt = samples.integrated.filt[,samples.integrated.filt$Immgen_sc_labels_agc=="Microglia"]
samples.integrated.filt <- RunUMAP(samples.integrated.filt, reduction = "pca", dims=1:15) %>%
FindNeighbors() %>% FindClusters(resolution = 0.6)
08:22:46 UMAP embedding parameters a = 0.9922 b = 1.112
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:22:46 Read 852 rows and found 15 numeric columns
08:22:46 Using Annoy for neighbor search, n_neighbors = 30
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by 'spam'
08:22:46 Building Annoy index with metric = cosine, n_trees = 50
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
08:22:47 Writing NN index file to temp file /tmp/RtmpajYTwQ/file15549c3059
08:22:47 Searching Annoy index using 1 thread, search_k = 3000
08:22:47 Annoy recall = 100%
08:22:48 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
08:22:50 Initializing from normalized Laplacian + noise (using RSpectra)
08:22:50 Commencing optimization for 500 epochs, with 33398 positive edges
08:22:52 Optimization finished
Computing nearest neighbor graph
Computing SNN
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 852
Number of edges: 26537
Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.8097
Number of communities: 7
Elapsed time: 0 seconds
n = length(unique(samples.integrated.filt$seurat_clusters))
samples.integrated.filt$labels<- factor(Idents(samples.integrated.filt),
levels = c(0:(n-1)), labels=paste0("Microglia", 0:(n-1))
)
Idents(samples.integrated.filt) = "labels"
a <- DimPlot(samples.integrated.filt, reduction="umap", label = T)
b <- DimPlot(samples.integrated.filt, reduction="umap", split.by = "Plate", label = F)
c <- DimPlot(samples.integrated.filt, reduction="umap", split.by = "Genotype_Treatment", label = F)
d <- DimPlot(samples.integrated.filt, reduction = "umap", group.by = "Genotype_Treatment")
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
c

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
d

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters.svg", a)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters_byPlate.svg", b)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Clusters_byCondition.svg", c)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Opt_All_genes_Condition.svg", d)
Saving 7 x 5 in image
Idents(samples.integrated.filt) <- "labels"
Markers=FindAllMarkers(samples.integrated.filt, logfc.threshold = 1.5)
Calculating cluster Microglia0
Calculating cluster Microglia1
Calculating cluster Microglia2
Calculating cluster Microglia3
Calculating cluster Microglia4
Calculating cluster Microglia5
Calculating cluster Microglia6
display_tab(Markers)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
Markers %>% dplyr::select(c("cluster", "gene")) %>% split(., "cluster") -> gogenelist
openxlsx::write.xlsx(Markers, "./output/Clusters_Markers_all.xlsx")
geneunivers<-rownames(samples.integrated.filt)
GOterm=getGOresults(gogenelist, genereference = geneunivers, organism = "mmusculus")
Loading required package: gprofiler2
display_tab(GOterm$result)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
openxlsx::write.xlsx(GOterm$result, "./output/Clusters_GOterms_Markers_all.xlsx")
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : P ISS ISO IEA,IM ... ,IMP,IBA,IMP ISO is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : I ISS ISO IBA IE ... IEA,IDA IBA IEA is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : sp90b1,Lpcat2,Nd ... p1,Dhrs7c,Wdyhv1 is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : O IBA IEA,ISO IB ... IEA,IDA IBA IEA is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : 2,Cryl1,Zfp541,S ... p1,Dhrs7c,Wdyhv1 is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : p5d,Papss1,Rnf7, ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : Mib2,Ilf3,Gpr146 ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : r84,Cds2,Gpr108, ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : s2,Gpr108,Rnf213 ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : Ccl12,Tmem50b,Ma ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : ,Plcl2,Rps14,Cct ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : ,Sap25,Map1a,Cap ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : Sf1,Prkcd,Smap2, ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : Sf1,Prkcd,Smap2, ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : l8,Pafah1b1,Rps1 ... 5,Gorasp1,Dhrs7c is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : it,Phf1,Trappc3, ... he,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : r2,Ppp1r10,Mrpl5 ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : a1d,Clec5a,Ppp1r ... 5,Gorasp1,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
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Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : grp3,Zscan26,Phf ... 15,Dhrs7c,Wdyhv1 is truncated.
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Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : 4,Vwde,Sec13,Cot ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : a6,Ormdl2,Rpl11, ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : 2,Patz1,Lrch3,Zd ... p1,Dhrs7c,Wdyhv1 is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : ,Maea,Nipsnap3b, ... 5,Gorasp1,Dhrs7c is truncated.
Number of characters exeed the limit of 32767.
Warning in wb$writeData(df = x, colNames = colNames, sheet = sheet, startCol = startCol, : ,Maea,Nipsnap3b, ... 5,Gorasp1,Dhrs7c is truncated.
Number of characters exeed the limit of 32767.
Idents(samples.integrated.filt) <- "labels"
Markers2vs4=FindMarkers(samples.integrated.filt,
ident.1 = "Microglia2", ident.2 = "Microglia4",
test.use = "LR", logfc.threshold = 1.5)
Warning: The following arguments are not used: norm.method
Warning: glm.fit: fitted probabilities numerically 0 or 1 occurred
Warning: glm.fit: fitted probabilities numerically 0 or 1 occurred
Warning: glm.fit: fitted probabilities numerically 0 or 1 occurred
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display_tab(Markers2vs4)
openxlsx::write.xlsx(Markers2vs4, "./output/Clusters_DEG_Micorglia2vs4.xlsx")
GOterm=getGOresults(list(Micorglia2vs4=rownames(Markers2vs4)[Markers2vs4$p_val_adj<0.05]), genereference = geneunivers, organism = "mmusculus")
display_tab(GOterm$result)
openxlsx::write.xlsx(GOterm$result, "./output/Clusters_GOterms_DEG_Micorglia2vs4.xlsx")
Idents(samples.integrated.filt) <- "labels"
Micorglia5vs6=FindMarkers(samples.integrated.filt,
ident.1 = "Microglia5",
ident.2 = "Microglia6",
test.use = "LR", logfc.threshold = 1.5)
Warning: The following arguments are not used: norm.method
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display_tab(Micorglia5vs6)
openxlsx::write.xlsx(Micorglia5vs6, "./output/Clusters_DEG_Markers2vs6.xlsx")
GOterm=getGOresults(geneset =list(Micorglia5vs6=rownames(Micorglia5vs6)[Micorglia5vs6$p_val_adj<0.05]),
genereference = geneunivers, organism = "mmusculus")
display_tab(GOterm$result)
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client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
openxlsx::write.xlsx(GOterm$result, "./output/Clusters_GOterms_DEG_Micorglia2vs6.xlsx")
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We will provide gene lists for different previously described microglia subclusters including homeostatic, DAM 1/DAM2 and IRM identification list. Please generate a Venn diagram or similar to see how big the overlap of our cells of interest (identified in the scDataset via the spatial transcriptomics list) is with the individual subclusters,
Profiles based on genelist provided by Jan Hoffmann
NOT done: Alterantive DAM profiles based on Cell. 2017 Jun 15;169(7):1276-1290.e17. doi: 10.1016/j.cell.2017.05.018)
#load("./data/DAM_genelists.RData")
DefaultAssay(samples.integrated.filt) = "SCT"
samples.integrated.filt.target<- AddModuleScore(object = samples.integrated.filt.target,
features = list(DAM1_up), name = "DAM_score")
#FeaturePlot(samples.integrated.filt.target, features = "DAM_score1")+scale_color_viridis_c()
samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(DAM_up), name = "DAM_score")
p4 <- FeaturePlot(object = samples.integrated.filt, features = "DAM_score1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
DAMcutoff = 1.5
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=DAM_score1))+
geom_density()+geom_vline(xintercept = DAMcutoff)+
theme_classic()
samples.integrated.filt$DAM_binary = as.factor(samples.integrated.filt$DAM_score1>=DAMcutoff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "DAM_binary")
comb = phist | p4 | pbin| p4a
comb

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
prop.table(table(DAMs=samples.integrated.filt$DAM_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
DAMs Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.01415094 0.73863636 1.00000000 0.66086957 0.97938144 0.94594595
TRUE 0.98584906 0.26136364 0.00000000 0.33913043 0.02061856 0.05405405
Clusters
DAMs Microglia6
FALSE 0.89655172
TRUE 0.10344828
dam_GT_p <-DimPlot(object = samples.integrated.filt, group.by = "DAM_binary", split.by = "Genotype_Treatment")
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM1_calling.svg", comb)
Saving 12 x 4 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM1_bygenotype.svg", dam_GT_p)
Saving 12 x 4 in image
phist

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
p4

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
pbin

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM1_thresholding.svg", phist)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM1_scoring.svg", p4)
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ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM1_binary.svg", pbin)
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samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(DAM2_up), name = "DAM2_score")
Warning: The following features are not present in the object: Lilrb4, not
searching for symbol synonyms
p4 <- FeaturePlot(object = samples.integrated.filt, features = "DAM2_score1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
DAM2cutoff = 1.0
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=DAM2_score1))+
geom_density()+geom_vline(xintercept = DAM2cutoff)+
theme_classic()
samples.integrated.filt$DAM2_binary = as.factor(samples.integrated.filt$DAM2_score1>=DAM2cutoff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "DAM2_binary")
comb = phist | p4 | pbin| p4a
dam_GT_p <-DimPlot(object = samples.integrated.filt, group.by = "DAM2_binary", split.by = "Genotype_Treatment")
comb

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
dam_GT_p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM2_calling.svg", comb)
Saving 12 x 4 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM2_bygenotype.svg", dam_GT_p)
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prop.table(table(DAM2=samples.integrated.filt$DAM2_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
DAM2 Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.108490566 0.914772727 0.991666667 0.834782609 0.989690722 0.945945946
TRUE 0.891509434 0.085227273 0.008333333 0.165217391 0.010309278 0.054054054
Clusters
DAM2 Microglia6
FALSE 0.913793103
TRUE 0.086206897
prop.table(table(DAM2=samples.integrated.filt$DAM2_binary, DAMs=samples.integrated.filt$DAM_binary), margin = 2)
DAMs
DAM2 FALSE TRUE
FALSE 0.994505495 0.245098039
TRUE 0.005494505 0.754901961
a <- ggplot(samples.integrated.filt@meta.data, aes(x=DAM2_score1, y=DAM_score1))+
geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("all cells")
b<- ggplot(samples.integrated.filt@meta.data[samples.integrated.filt$DAM_binary==TRUE,], aes(x=DAM2_score1, y=DAM_score1))+geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("DAMs only")
combo <- a|b
combo

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAMvsDAM2_contourplot.svg", combo)
Saving 12 x 5 in image
proportion tables show percebtages of DAM cells per condition
metadata=samples.integrated.filt@meta.data
restab = table(GT_Treat = metadata$Genotype_Treatment, DAMs=metadata$DAM_binary)
restab
DAMs
GT_Treat FALSE TRUE
WT_Ctrl 235 1
WT_Stroke 65 61
APPPS1_Ctrl 134 169
APPPS1_Stroke 112 75
prop.table(restab, margin = 1)
DAMs
GT_Treat FALSE TRUE
WT_Ctrl 0.995762712 0.004237288
WT_Stroke 0.515873016 0.484126984
APPPS1_Ctrl 0.442244224 0.557755776
APPPS1_Stroke 0.598930481 0.401069519
chisq.test(restab)
Pearson's Chi-squared test
data: restab
X-squared = 191.06, df = 3, p-value < 2.2e-16
proplot <- ggplot(as.data.frame(restab),
aes(fill=DAMs, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()+ggtitle("DAM proportions")
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAMproportions_per_condition_all.svg", proplot)
Saving 7 x 5 in image
library(chisq.posthoc.test)
chisq.posthoc.test(restab)
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals 13.365819 -13.365819
2 WT_Ctrl p values 0.000000 0.000000
3 WT_Stroke Residuals -3.167614 3.167614
4 WT_Stroke p values 0.012296 0.012296
5 APPPS1_Ctrl Residuals -8.976739 8.976739
6 APPPS1_Ctrl p values 0.000000 0.000000
7 APPPS1_Stroke Residuals -1.352314 1.352314
8 APPPS1_Stroke p values 1.000000 1.000000
#To Do Pairwise
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "APPPS1", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), DAMs=metadata$DAM_binary)
restab
DAMs
GT_Treat FALSE TRUE
APPPS1_Ctrl 134 169
APPPS1_Stroke 112 75
prop.table(restab, margin = 1)
DAMs
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.4422442 0.5577558
APPPS1_Stroke 0.5989305 0.4010695
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 0.0008179
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.3602999 0.7815525
sample estimates:
odds ratio
0.5316483
proplot<-ggplot(as.data.frame(restab),
aes(fill=DAMs, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAMproportions_per_condition_APPPS1.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "WT", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), DAMs=metadata$DAM_binary)
restab
DAMs
GT_Treat FALSE TRUE
WT_Ctrl 235 1
WT_Stroke 65 61
prop.table(restab, margin = 1)
DAMs
GT_Treat FALSE TRUE
WT_Ctrl 0.995762712 0.004237288
WT_Stroke 0.515873016 0.484126984
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value < 2.2e-16
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
36.09295 8376.89342
sample estimates:
odds ratio
216.2896
proplot<-ggplot(as.data.frame(restab),
aes(fill=DAMs, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAMproportions_per_condition_WT.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$DAM_binary==TRUE,]
restab = table(GT_Treat = metadata$Genotype_Treatment, DAM2=metadata$DAM2_binary)
restab
DAM2
GT_Treat FALSE TRUE
WT_Ctrl 0 1
WT_Stroke 38 23
APPPS1_Ctrl 15 154
APPPS1_Stroke 22 53
prop.table(restab, margin = 1)
DAM2
GT_Treat FALSE TRUE
WT_Ctrl 0.0000000 1.0000000
WT_Stroke 0.6229508 0.3770492
APPPS1_Ctrl 0.0887574 0.9112426
APPPS1_Stroke 0.2933333 0.7066667
chisq.test(restab)
Warning in chisq.test(restab): Chi-squared approximation may be incorrect
Pearson's Chi-squared test
data: restab
X-squared = 70.663, df = 3, p-value = 3.078e-15
proplot<-ggplot(as.data.frame(restab),
aes(fill=DAM2, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
chisq.posthoc.test(restab)
Warning in chisq.test(x, ...): Chi-squared approximation may be incorrect
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals -0.5707362 0.5707362
2 WT_Ctrl p values 1.0000000 1.0000000
3 WT_Stroke Residuals 7.6674335 -7.6674335
4 WT_Stroke p values 0.0000000 0.0000000
5 APPPS1_Ctrl Residuals -7.0615572 7.0615572
6 APPPS1_Ctrl p values 0.0000000 0.0000000
7 APPPS1_Stroke Residuals 1.1177253 -1.1177253
8 APPPS1_Stroke p values 1.0000000 1.0000000
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM2proportionsofDAM1_per_condition.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[(samples.integrated.filt$DAM_binary==TRUE) & (samples.integrated.filt$Genotype=="APPPS1"),]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), DAM2=metadata$DAM2_binary)
restab
DAM2
GT_Treat FALSE TRUE
APPPS1_Ctrl 15 154
APPPS1_Stroke 22 53
prop.table(restab, margin = 1)
DAM2
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.0887574 0.9112426
APPPS1_Stroke 0.2933333 0.7066667
chisq.test(restab)
Pearson's Chi-squared test with Yates' continuity correction
data: restab
X-squared = 15.347, df = 1, p-value = 8.948e-05
proplot<- ggplot(as.data.frame(restab),
aes(fill=DAM2, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM2proportionsofDAM1_per_condition_APPPS1.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[(samples.integrated.filt$DAM_binary==TRUE) & (samples.integrated.filt$Genotype=="WT"),]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), DAM2=metadata$DAM2_binary)
restab
DAM2
GT_Treat FALSE TRUE
WT_Ctrl 0 1
WT_Stroke 38 23
prop.table(restab, margin = 1)
DAM2
GT_Treat FALSE TRUE
WT_Ctrl 0.0000000 1.0000000
WT_Stroke 0.6229508 0.3770492
chisq.test(restab)
Warning in chisq.test(restab): Chi-squared approximation may be incorrect
Pearson's Chi-squared test with Yates' continuity correction
data: restab
X-squared = 0.054609, df = 1, p-value = 0.8152
proplot<-ggplot(as.data.frame(restab),
aes(fill=DAM2, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAM2proportionsofDAM1_per_condition_WT.svg", proplot)
Saving 7 x 5 in image
# list from Jan Hofmann
a <- RidgePlot(samples.integrated.filt, features = homeostat.genes, ncol = 2)
a
Picking joint bandwidth of 0.274
Picking joint bandwidth of 0.462
Picking joint bandwidth of 0.327
Picking joint bandwidth of 0.329
Picking joint bandwidth of 0.333
Picking joint bandwidth of 0.491

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
b<- FeaturePlot(samples.integrated.filt, features = homeostat.genes)
b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_HomeostatGenes_Cluster_Ridgeplot.svg", a)
Saving 8 x 15 in image
Picking joint bandwidth of 0.274
Picking joint bandwidth of 0.462
Picking joint bandwidth of 0.327
Picking joint bandwidth of 0.329
Picking joint bandwidth of 0.333
Picking joint bandwidth of 0.491
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_HomeostatGenes_Featureplot.svg", a)
Saving 8 x 15 in image
Picking joint bandwidth of 0.274
Picking joint bandwidth of 0.462
Picking joint bandwidth of 0.327
Picking joint bandwidth of 0.329
Picking joint bandwidth of 0.333
Picking joint bandwidth of 0.491
samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(homeostat.genes), name = "Homeo_score")
p4 <- FeaturePlot(object = samples.integrated.filt, features = "Homeo_score1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
Homeocutff = 2
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=Homeo_score1))+
geom_density()+geom_vline(xintercept = Homeocutff)+theme_classic()
samples.integrated.filt$Homeo_binary = as.factor(samples.integrated.filt$Homeo_score1>=Homeocutff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "Homeo_binary")
comb = phist | p4 | pbin| p4a
Hom_GT_p <-DimPlot(object = samples.integrated.filt, group.by = "Homeo_binary", split.by = "Genotype_Treatment")
comb

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
Hom_GT_p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
prop.table(table(Homeo=samples.integrated.filt$Homeo_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
Homeo Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.94339623 0.30113636 0.03333333 0.48695652 0.18556701 0.85135135
TRUE 0.05660377 0.69886364 0.96666667 0.51304348 0.81443299 0.14864865
Clusters
Homeo Microglia6
FALSE 0.96551724
TRUE 0.03448276
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_calling.svg", comb)
Saving 12 x 4 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_bygenotype.svg", Hom_GT_p)
Saving 12 x 4 in image
phist

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
p4

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
pbin

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_thresholding.svg", phist)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_scoring.svg", p4)
Saving 7 x 5 in image
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_binary.svg", pbin)
Saving 7 x 5 in image
metadata = samples.integrated.filt@meta.data
metadata$Homeo_DAM <- factor(1*as.logical(metadata$Homeo_binary) + 2*as.logical(metadata$DAM_binary), levels = c(0,1,2,3), labels = c("Neither", "Homeo", "DAM", "Unknown"))
samples.integrated.filt@meta.data <- metadata
a = DimPlot(samples.integrated.filt, reduction="umap", group.by = "Homeo_DAM")
a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeo_DAM.svg", a)
Saving 7 x 5 in image
a <- ggplot(samples.integrated.filt@meta.data, aes(x=Homeo_score1, y=DAM_score1))+
geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("all cells")
b<- ggplot(samples.integrated.filt@meta.data[samples.integrated.filt$DAM_binary==TRUE,], aes(x=Homeo_score1, y=DAM_score1))+geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("DAMs only")
a|b

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_DAMvsHomeo.svg", a|b)
Saving 12 x 5 in image
proportion tables show percentages of Homeostatic cells per condition
metadata=samples.integrated.filt@meta.data
restab = table(GT_Treat = metadata$Genotype_Treatment, Homeo=metadata$Homeo_binary)
restab
Homeo
GT_Treat FALSE TRUE
WT_Ctrl 21 215
WT_Stroke 78 48
APPPS1_Ctrl 186 117
APPPS1_Stroke 165 22
prop.table(restab, margin = 1)
Homeo
GT_Treat FALSE TRUE
WT_Ctrl 0.08898305 0.91101695
WT_Stroke 0.61904762 0.38095238
APPPS1_Ctrl 0.61386139 0.38613861
APPPS1_Stroke 0.88235294 0.11764706
chisq.test(restab)
Pearson's Chi-squared test
data: restab
X-squared = 289.9, df = 3, p-value < 2.2e-16
proplot<-ggplot(as.data.frame(restab),
aes(fill=Homeo, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
chisq.posthoc.test(restab)
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals -15.894768 15.894768
2 WT_Ctrl p values 0.000000 0.000000
3 WT_Stroke Residuals 2.213700 -2.213700
4 WT_Stroke p values 0.214796 0.214796
5 APPPS1_Ctrl Residuals 3.722352 -3.722352
6 APPPS1_Ctrl p values 0.001579 0.001579
7 APPPS1_Stroke Residuals 10.981937 -10.981937
8 APPPS1_Stroke p values 0.000000 0.000000
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeoproportions_per_condition.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype=="APPPS1",]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Homeo=metadata$Homeo_binary)
restab
Homeo
GT_Treat FALSE TRUE
APPPS1_Ctrl 186 117
APPPS1_Stroke 165 22
prop.table(restab, margin = 1)
Homeo
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.6138614 0.3861386
APPPS1_Stroke 0.8823529 0.1176471
chisq.test(restab)
Pearson's Chi-squared test with Yates' continuity correction
data: restab
X-squared = 39.712, df = 1, p-value = 2.944e-10
proplot<- ggplot(as.data.frame(restab),
aes(fill=Homeo, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeoproportions_per_condition_APPPS1.svg", proplot)
Saving 7 x 5 in image
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype=="WT",]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Homeo=metadata$Homeo_binary)
restab
Homeo
GT_Treat FALSE TRUE
WT_Ctrl 21 215
WT_Stroke 78 48
prop.table(restab, margin = 1)
Homeo
GT_Treat FALSE TRUE
WT_Ctrl 0.08898305 0.91101695
WT_Stroke 0.61904762 0.38095238
chisq.test(restab)
Pearson's Chi-squared test with Yates' continuity correction
data: restab
X-squared = 113.51, df = 1, p-value < 2.2e-16
proplot<-ggplot(as.data.frame(restab),
aes(fill=Homeo, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()
proplot

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Homeoproportions_per_condition_WT.svg", proplot)
Saving 7 x 5 in image
### Spatial cell types
Idents(samples.integrated.filt) = "Genotype_Treatment"
DoHeatmap(samples.integrated.filt, assay = "RNA", features = c(distal_genes, control_genes, Stroke_genes))+ggtitle("spatial genes all")
Warning in DoHeatmap(samples.integrated.filt, assay = "RNA", features =
c(distal_genes, : The following features were omitted as they were not found in
the scale.data slot for the RNA assay: Timp1, Tmsb4x, Gm14539, Cx3cl1, Gm15564,
Norad, Atp5md, Bex1, Nrsn1, H2az1, Prxl2b, Stmn3, Gng13

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
DoHeatmap(samples.integrated.filt, assay = "RNA",features = c(distal_genes))+ggtitle("spatial genes distal")
Warning in DoHeatmap(samples.integrated.filt, assay = "RNA", features =
c(distal_genes)): The following features were omitted as they were not found in
the scale.data slot for the RNA assay: Bex1, Nrsn1, H2az1, Prxl2b, Stmn3, Gng13

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
DoHeatmap(samples.integrated.filt, assay = "RNA",features = c(control_genes))+ggtitle("spatial genes control")
Warning in DoHeatmap(samples.integrated.filt, assay = "RNA", features =
c(control_genes)): The following features were omitted as they were not found
in the scale.data slot for the RNA assay: Gm14539, Cx3cl1, Gm15564, Norad,
Atp5md

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
DoHeatmap(samples.integrated.filt, assay = "RNA",features = c(Stroke_genes))+ggtitle("spatial genes peri-ictus")
Warning in DoHeatmap(samples.integrated.filt, assay = "RNA", features =
c(Stroke_genes)): The following features were omitted as they were not found in
the scale.data slot for the RNA assay: Timp1, Tmsb4x

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
#load("./data/DAM_genelists.RData")
DefaultAssay(samples.integrated.filt) = "SCT"
samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(distal_genes), name = "Distal")
Warning: The following features are not present in the object: Gng13, Stmn3,
Prxl2b, H2az1, Ctxn1, Nrsn1, Bex1, Nrgn, Cck, not searching for symbol synonyms
p4 <- FeaturePlot(object = samples.integrated.filt, features = "Distal1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
Distalcutoff = 0.5
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=Distal1))+
geom_density()+geom_vline(xintercept = Distalcutoff)+
theme_classic()
samples.integrated.filt$Distal_binary = as.factor(samples.integrated.filt$Distal1>=Distalcutoff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "Distal_binary")
phist | p4 | pbin| p4a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
prop.table(table(Distals=samples.integrated.filt$Distal_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
Distals Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.39150943 0.89204545 0.97500000 0.91304348 0.97938144 1.00000000
TRUE 0.60849057 0.10795455 0.02500000 0.08695652 0.02061856 0.00000000
Clusters
Distals Microglia6
FALSE 1.00000000
TRUE 0.00000000
DimPlot(object = samples.integrated.filt, group.by = "Distal_binary", split.by = "Genotype_Treatment")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
#load("./data/DAM_genelists.RData")
DefaultAssay(samples.integrated.filt) = "SCT"
samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(control_genes), name = "Control")
Warning: The following features are not present in the object: Atp5md, Nrgn,
Norad, Dnm1, Gm15564, Cx3cl1, Gm14539, not searching for symbol synonyms
p4 <- FeaturePlot(object = samples.integrated.filt, features = "Control1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
Controlcutoff = 0.25
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=Control1))+
geom_density()+geom_vline(xintercept = Controlcutoff)+
theme_classic()
samples.integrated.filt$Control_binary = as.factor(samples.integrated.filt$Control1>=Controlcutoff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "Control_binary")
phist | p4 | pbin| p4a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
prop.table(table(Controls=samples.integrated.filt$Control_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
Controls Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.7075472 0.6590909 0.5750000 0.7565217 0.5979381 0.8783784
TRUE 0.2924528 0.3409091 0.4250000 0.2434783 0.4020619 0.1216216
Clusters
Controls Microglia6
FALSE 0.8965517
TRUE 0.1034483
DimPlot(object = samples.integrated.filt, group.by = "Control_binary", split.by = "Genotype_Treatment")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
#load("./data/DAM_genelists.RData")
DefaultAssay(samples.integrated.filt) = "SCT"
samples.integrated.filt <- AddModuleScore(object = samples.integrated.filt,
features = list(Stroke_genes), name = "Peri_ictus")
Warning: The following features are not present in the object: Serpina3n,
Tmsb4x, Timp1, not searching for symbol synonyms
p4 <- FeaturePlot(object = samples.integrated.filt, features = "Peri_ictus1")+scale_color_viridis_c()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p4a <- DimPlot(object = samples.integrated.filt, reduction = "umap")
Strokecutoff = 2.5
phist = ggplot(data = samples.integrated.filt@meta.data, aes(x=Peri_ictus1))+
geom_density()+geom_vline(xintercept = Strokecutoff)+
theme_classic()+ggtitle("Peri_ictus")
samples.integrated.filt$Peri_ictus_binary = as.factor(samples.integrated.filt$Peri_ictus1>=Strokecutoff)
pbin <- DimPlot(object = samples.integrated.filt, group.by = "Peri_ictus_binary")
phist | p4 | pbin| p4a

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
prop.table(table(Strokes=samples.integrated.filt$Peri_ictus_binary, Clusters=samples.integrated.filt$labels), margin = 2)
Clusters
Strokes Microglia0 Microglia1 Microglia2 Microglia3 Microglia4 Microglia5
FALSE 0.089622642 0.710227273 0.991666667 0.747826087 1.000000000 0.959459459
TRUE 0.910377358 0.289772727 0.008333333 0.252173913 0.000000000 0.040540541
Clusters
Strokes Microglia6
FALSE 0.982758621
TRUE 0.017241379
DimPlot(object = samples.integrated.filt, group.by = "Peri_ictus_binary", split.by = "Genotype_Treatment")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggplot(samples.integrated.filt@meta.data, aes(x=Peri_ictus1, y=Control1))+
geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("all cells")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggplot(samples.integrated.filt@meta.data[samples.integrated.filt$Peri_ictus_binary==TRUE,], aes(x=Peri_ictus1, y=Control1))+geom_density_2d_filled()+geom_point(col="#FFFFFF55")+theme_classic()+ggtitle("Peri_ictus only")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
proportion tables show percebtages of DAM cells per condition
metadata=samples.integrated.filt@meta.data
restab = table(GT_Treat = metadata$Genotype_Treatment, Distals=metadata$Distal_binary)
restab
Distals
GT_Treat FALSE TRUE
WT_Ctrl 229 7
WT_Stroke 110 16
APPPS1_Ctrl 195 108
APPPS1_Stroke 155 32
prop.table(restab, margin = 1)
Distals
GT_Treat FALSE TRUE
WT_Ctrl 0.97033898 0.02966102
WT_Stroke 0.87301587 0.12698413
APPPS1_Ctrl 0.64356436 0.35643564
APPPS1_Stroke 0.82887701 0.17112299
chisq.test(restab)
Pearson's Chi-squared test
data: restab
X-squared = 97.122, df = 3, p-value < 2.2e-16
ggplot(as.data.frame(restab),
aes(fill=Distals, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
library(chisq.posthoc.test)
chisq.posthoc.test(restab)
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals 7.4251774 -7.4251774
2 WT_Ctrl p values 0.0000000 0.0000000
3 WT_Stroke Residuals 1.9887921 -1.9887921
4 WT_Stroke p values 0.3737930 0.3737930
5 APPPS1_Ctrl Residuals -9.1031912 9.1031912
6 APPPS1_Ctrl p values 0.0000000 0.0000000
7 APPPS1_Stroke Residuals 0.7945767 -0.7945767
8 APPPS1_Stroke p values 1.0000000 1.0000000
#To Do Pairwise
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "APPPS1", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Distals=metadata$Distal_binary)
restab
Distals
GT_Treat FALSE TRUE
APPPS1_Ctrl 195 108
APPPS1_Stroke 155 32
prop.table(restab, margin = 1)
Distals
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.6435644 0.3564356
APPPS1_Stroke 0.8288770 0.1711230
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 8.579e-06
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.2304338 0.5934206
sample estimates:
odds ratio
0.3734791
ggplot(as.data.frame(restab),
aes(fill=Distals, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "WT", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Distals=metadata$Distal_binary)
restab
Distals
GT_Treat FALSE TRUE
WT_Ctrl 229 7
WT_Stroke 110 16
prop.table(restab, margin = 1)
Distals
GT_Treat FALSE TRUE
WT_Ctrl 0.97033898 0.02966102
WT_Stroke 0.87301587 0.12698413
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 0.0005206
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
1.780926 14.027864
sample estimates:
odds ratio
4.736223
ggplot(as.data.frame(restab),
aes(fill=Distals, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
metadata=samples.integrated.filt@meta.data
restab = table(GT_Treat = metadata$Genotype_Treatment, Controls=metadata$Control_binary)
restab
Controls
GT_Treat FALSE TRUE
WT_Ctrl 142 94
WT_Stroke 90 36
APPPS1_Ctrl 223 80
APPPS1_Stroke 142 45
prop.table(restab, margin = 1)
Controls
GT_Treat FALSE TRUE
WT_Ctrl 0.6016949 0.3983051
WT_Stroke 0.7142857 0.2857143
APPPS1_Ctrl 0.7359736 0.2640264
APPPS1_Stroke 0.7593583 0.2406417
chisq.test(restab)
Pearson's Chi-squared test
data: restab
X-squared = 16.007, df = 3, p-value = 0.00113
ggplot(as.data.frame(restab),
aes(fill=Controls, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
library(chisq.posthoc.test)
chisq.posthoc.test(restab)
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals -3.9061041 3.9061041
2 WT_Ctrl p values 0.0007500 0.0007500
3 WT_Stroke Residuals 0.3606338 -0.3606338
4 WT_Stroke p values 1.0000000 1.0000000
5 APPPS1_Ctrl Residuals 1.6700713 -1.6700713
6 APPPS1_Ctrl p values 0.7592420 0.7592420
7 APPPS1_Stroke Residuals 1.9824864 -1.9824864
8 APPPS1_Stroke p values 0.3793990 0.3793990
#To Do Pairwise
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "APPPS1", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Controls=metadata$Control_binary)
restab
Controls
GT_Treat FALSE TRUE
APPPS1_Ctrl 223 80
APPPS1_Stroke 142 45
prop.table(restab, margin = 1)
Controls
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.7359736 0.2640264
APPPS1_Stroke 0.7593583 0.2406417
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 0.5949
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.5645775 1.3721440
sample estimates:
odds ratio
0.8835869
ggplot(as.data.frame(restab),
aes(fill=Controls, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "WT", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Controls=metadata$Control_binary)
restab
Controls
GT_Treat FALSE TRUE
WT_Ctrl 142 94
WT_Stroke 90 36
prop.table(restab, margin = 1)
Controls
GT_Treat FALSE TRUE
WT_Ctrl 0.6016949 0.3983051
WT_Stroke 0.7142857 0.2857143
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 0.03847
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.3671052 0.9850947
sample estimates:
odds ratio
0.6050892
ggplot(as.data.frame(restab),
aes(fill=Controls, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
metadata=samples.integrated.filt@meta.data
restab = table(GT_Treat = metadata$Genotype_Treatment, Peri_Ictal=metadata$Peri_ictus_binary)
restab
Peri_Ictal
GT_Treat FALSE TRUE
WT_Ctrl 235 1
WT_Stroke 59 67
APPPS1_Ctrl 151 152
APPPS1_Stroke 129 58
prop.table(restab, margin = 1)
Peri_Ictal
GT_Treat FALSE TRUE
WT_Ctrl 0.995762712 0.004237288
WT_Stroke 0.468253968 0.531746032
APPPS1_Ctrl 0.498349835 0.501650165
APPPS1_Stroke 0.689839572 0.310160428
chisq.test(restab)
Pearson's Chi-squared test
data: restab
X-squared = 178.15, df = 3, p-value < 2.2e-16
ggplot(as.data.frame(restab),
aes(fill=Peri_Ictal, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
library(chisq.posthoc.test)
chisq.posthoc.test(restab)
Dimension Value FALSE TRUE
1 WT_Ctrl Residuals 12.4100951 -12.4100951
2 WT_Ctrl p values 0.0000000 0.0000000
3 WT_Stroke Residuals -5.3286184 5.3286184
4 WT_Stroke p values 0.0000010 0.0000010
5 APPPS1_Ctrl Residuals -8.1104408 8.1104408
6 APPPS1_Ctrl p values 0.0000000 0.0000000
7 APPPS1_Stroke Residuals 0.5325286 -0.5325286
8 APPPS1_Stroke p values 1.0000000 1.0000000
#To Do Pairwise
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "APPPS1", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Peri_Ictal=metadata$Peri_ictus_binary)
restab
Peri_Ictal
GT_Treat FALSE TRUE
APPPS1_Ctrl 151 152
APPPS1_Stroke 129 58
prop.table(restab, margin = 1)
Peri_Ictal
GT_Treat FALSE TRUE
APPPS1_Ctrl 0.4983498 0.5016502
APPPS1_Stroke 0.6898396 0.3101604
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value = 3.416e-05
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.2983945 0.6660541
sample estimates:
odds ratio
0.4473919
ggplot(as.data.frame(restab),
aes(fill=Peri_Ictal, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
metadata=samples.integrated.filt@meta.data[samples.integrated.filt$Genotype == "WT", ]
restab = table(GT_Treat = as.character(metadata$Genotype_Treatment), Peri_Ictal=metadata$Peri_ictus_binary)
restab
Peri_Ictal
GT_Treat FALSE TRUE
WT_Ctrl 235 1
WT_Stroke 59 67
prop.table(restab, margin = 1)
Peri_Ictal
GT_Treat FALSE TRUE
WT_Ctrl 0.995762712 0.004237288
WT_Stroke 0.468253968 0.531746032
fisher.test(restab)
Fisher's Exact Test for Count Data
data: restab
p-value < 2.2e-16
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
43.61937 9976.27448
sample estimates:
odds ratio
261.1922
ggplot(as.data.frame(restab),
aes(fill=Peri_Ictal, x=GT_Treat, y=Freq))+
geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
library(scSorter)
annodf = data.frame(Type = "Periictus", Marker = Stroke_genes)
annodf = rbind(annodf, data.frame(Type = "Control", Marker = control_genes))
annodf = rbind(annodf, data.frame(Type = "Distal", Marker = distal_genes))
spatialtypes = scSorter(samples.integrated.filt@assays$SCT@counts, annodf) #makes the calling
Warning in data_preprocess(expr, anno_processed): The following specified
marker genes are not found from the expression data: SERPINA3N, TMSB4X, TIMP1,
ATP5MD, NRGN, NORAD, DNM1, GM15564, CX3CL1, GM14539, GNG13, STMN3, PRXL2B,
H2AZ1, CTXN1, NRSN1, BEX1, CCK.
metadata = samples.integrated.filt@meta.data
metadata = metadata[,! colnames(metadata) %in% c("PredType")]
samples.integrated.filt@meta.data <- cbind(metadata, PredType = spatialtypes$Pred_Type)
DimPlot(samples.integrated.filt, group.by = "PredType", split.by = "Genotype_Treatment")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
samples.integrated.filt.comorbid <- samples.integrated.filt[,metadata$Genotype_Treatment == "APPPS1_Stroke"]
DimPlot(samples.integrated.filt.comorbid, group.by = "PredType", )

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
crosstab = table(Condition = samples.integrated.filt$Genotype_Treatment, Spatial_Type = samples.integrated.filt$PredType)
ggplot(as.data.frame(crosstab), aes(fill=Spatial_Type, x=Condition, y=Freq)) + geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
crosstab
Spatial_Type
Condition Control Distal Periictus Unknown
WT_Ctrl 12 0 221 3
WT_Stroke 13 3 108 2
APPPS1_Ctrl 10 0 292 1
APPPS1_Stroke 46 14 113 14
chisq.test(crosstab)
Warning in chisq.test(crosstab): Chi-squared approximation may be incorrect
Pearson's Chi-squared test
data: crosstab
X-squared = 148.47, df = 9, p-value < 2.2e-16
ggplot(as.data.frame(crosstab[1:2,]), aes(fill=Spatial_Type, x=Condition, y=Freq)) + geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
crosstab[1:2,]
Spatial_Type
Condition Control Distal Periictus Unknown
WT_Ctrl 12 0 221 3
WT_Stroke 13 3 108 2
chisq.test(crosstab[1:2,])
Warning in chisq.test(crosstab[1:2, ]): Chi-squared approximation may be
incorrect
Pearson's Chi-squared test
data: crosstab[1:2, ]
X-squared = 9.5037, df = 3, p-value = 0.02329
ggplot(as.data.frame(crosstab[3:4,]), aes(fill=Spatial_Type, x=Condition, y=Freq)) + geom_bar(position="fill", stat="identity")+theme_classic()

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
crosstab[3:4,]
Spatial_Type
Condition Control Distal Periictus Unknown
APPPS1_Ctrl 10 0 292 1
APPPS1_Stroke 46 14 113 14
chisq.test(crosstab[3:4,])
Pearson's Chi-squared test
data: crosstab[3:4, ]
X-squared = 106, df = 3, p-value < 2.2e-16
avg_logFC: log fold-chage of the average expression between the two groups. Positive values indicate that the gene is more highly expressed in the first group
Idents(samples.integrated.filt) <- "Genotype_Treatment"
DimPlot(samples.integrated.filt)

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
DEG_Wt_CtrlvsStroke <-FindMarkers(samples.integrated.filt,
ident.1 = "WT_Stroke", ident.2 = "WT_Ctrl",
test.use = "LR", logfc.threshold = 0)
Warning: The following arguments are not used: norm.method
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p = EnhancedVolcano::EnhancedVolcano(DEG_Wt_CtrlvsStroke, x = "avg_log2FC",
y = "p_val_adj", lab=rownames(DEG_Wt_CtrlvsStroke), title = "WT_Ctrl vs WT_Stroke")
display_tab(DEG_Wt_CtrlvsStroke)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Volcano_WTCtrlvsWTStroke.svg", p)
Saving 6 x 6 in image
avg_logFC: log fold-chage of the average expression between the two groups. Positive values indicate that the gene is more highly expressed in the first group
DEG_APPPS1_CtrlvsStroke <-FindMarkers(samples.integrated.filt,
ident.1 = "APPPS1_Stroke", ident.2 = "APPPS1_Ctrl",
test.use = "LR", logfc.threshold = 0)
Warning: The following arguments are not used: norm.method
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display_tab(DEG_APPPS1_CtrlvsStroke)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
p<- EnhancedVolcano::EnhancedVolcano(DEG_APPPS1_CtrlvsStroke, x = "avg_log2FC", y = "p_val_adj",
lab=rownames(DEG_APPPS1_CtrlvsStroke), title = "APPPS1_Ctrl vs APPPS1_Stroke")
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Volcano_APPPS1CtrlvsAPPPS1Stroke.svg", p)
Saving 6 x 6 in image
DEG_Stroke_APPPS1vsWT <-FindMarkers(samples.integrated.filt,
ident.1 = "APPPS1_Stroke", ident.2 = "WT_Stroke",
test.use = "LR", logfc.threshold = 0)
Warning: The following arguments are not used: norm.method
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display_tab(DEG_Stroke_APPPS1vsWT)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
p<- EnhancedVolcano::EnhancedVolcano(DEG_Stroke_APPPS1vsWT, x = "avg_log2FC", y = "p_val_adj",
lab=rownames(DEG_Stroke_APPPS1vsWT), title = "WT_stroke vs APPPS1_Stroke")
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Volcano_WTstrokevsAPPPS1Stroke.svg", p)
Saving 6 x 6 in image
avg_logFC: log fold-chage of the average expression between the two groups. Positive values indicate that the gene is more highly expressed in the first group
DEG_APPPS1_CtrlvsStroke <-FindMarkers(samples.integrated.filt,
ident.1 = "APPPS1_Ctrl", ident.2 = "WT_Ctrl",
test.use = "LR", logfc.threshold = 0)
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display_tab(DEG_APPPS1_CtrlvsStroke)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
p<- EnhancedVolcano::EnhancedVolcano(DEG_APPPS1_CtrlvsStroke, x = "avg_log2FC", y = "p_val_adj",
lab=rownames(DEG_APPPS1_CtrlvsStroke), title = "WT_Ctrl vs APPPS1_Ctrl")
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Volcano_WTCtrlvsAPPPS1ctrl.svg", p)
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genelist_both= intersect(rownames(DEG_Wt_CtrlvsStroke), rownames(DEG_APPPS1_CtrlvsStroke))
plotdata=data.frame(log2FC_WTCtrl_vs_WTStroke=DEG_Wt_CtrlvsStroke[genelist_both,]$avg_log2FC, log2FC_APPPS1Ctrl_vs_APPPS1Stroke = DEG_APPPS1_CtrlvsStroke[genelist_both,]$avg_log2FC)
rownames(plotdata) = genelist_both
plotdata$label = rownames(plotdata)
n=20
plotdata$label[! c(1:nrow(plotdata)) %in%
c(
order(abs(plotdata$log2FC_WTCtrl_vs_WTStroke), decreasing = T)[1:n],
order(abs(plotdata$log2FC_APPPS1Ctrl_vs_APPPS1Stroke), decreasing = T)[1:n])]=""
# plotdata$col="WT"
# apps1=abs(plotdata$APPPS1)>abs(plotdata$WT)
# plotdata$col[ apps1]="APPPS1"
# plotdata$col[(abs(plotdata$APPPS1)<0.25) & (abs(plotdata$WT)<0.25)] <- NA
p <- ggplot(plotdata, aes(x=log2FC_WTCtrl_vs_WTStroke, y=log2FC_APPPS1Ctrl_vs_APPPS1Stroke, label=label))+geom_point(alpha=0.7)+
geom_text(vjust=-0.7, color="black")+
#geom_abline(slope=-1, intercept=0, lty=2)+geom_abline(slope=1, intercept=0, lty=2)+
theme_classic()+ggtitle("log_FC")
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_logFCCtrlvsStroke_APPPS1vsWT_all.svg", p)
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# sig only
genelist_both= unique(c(rownames(DEG_Wt_CtrlvsStroke[DEG_Wt_CtrlvsStroke$p_val<0.05,]), rownames(DEG_APPPS1_CtrlvsStroke[DEG_APPPS1_CtrlvsStroke$p_val>0.05,])))
plotdata=data.frame(log2FC_WTCtrl_vs_WTStroke=DEG_Wt_CtrlvsStroke[genelist_both,]$avg_log2FC, log2FC_APPPS1Ctrl_vs_APPPS1Stroke = DEG_APPPS1_CtrlvsStroke[genelist_both,]$avg_log2FC)
rownames(plotdata) = genelist_both
plotdata$label = rownames(plotdata)
n=15
plotdata$label[! c(1:nrow(plotdata)) %in%
c(
order(abs(plotdata$log2FC_WTCtrl_vs_WTStroke), decreasing = T)[1:n],
order(abs(plotdata$log2FC_APPPS1Ctrl_vs_APPPS1Stroke), decreasing = T)[1:n])]=""
# plotdata$col="WT"
# apps1=abs(plotdata$APPPS1)>abs(plotdata$WT)
# plotdata$col[ apps1]="APPPS1"
# plotdata$col[(abs(plotdata$APPPS1)<0.25) & (abs(plotdata$WT)<0.25)] <- NA
p <- ggplot(plotdata, aes(x=log2FC_WTCtrl_vs_WTStroke, y=log2FC_APPPS1Ctrl_vs_APPPS1Stroke, label=label))+geom_point(alpha=0.7)+
geom_text(vjust=-0.7, color="black")+
#geom_abline(slope=-1, intercept=0, lty=2)+geom_abline(slope=1, intercept=0, lty=2)+
theme_classic()+ggtitle("log_FC")
p
Warning: Removed 301 rows containing missing values (`geom_point()`).
Warning: Removed 301 rows containing missing values (`geom_text()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_logFCCtrlvsStroke_APPPS1vsWT_sig_only.svg", p)
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reads_data = samples.integrated.filt@assays$RNA@scale.data
barplot_genes = function(gene, split.by.cluster =T){
library(ggbeeswarm)
plotdata = data.frame(log2Reads = log2(reads_data[gene,]+1),
Genotype_Treatment = samples.integrated.filt@meta.data$Genotype_Treatment,
Cluster = samples.integrated.filt@meta.data$labels)
p=ggplot(plotdata, aes(x=Genotype_Treatment, y=log2Reads, fill=Genotype_Treatment))+geom_boxplot()+geom_beeswarm(alpha=0.4)+ggtitle(gene)+theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))
if(split.by.cluster){p=p+facet_wrap(~Cluster)}
p
}
barplot_genes(gene="Spp1", split.by.cluster =T)

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
DEG_Strokebins_Wt_CtrlvsStroke <-FindMarkers(samples.integrated.filt, subset.ident = "Stroke_bin", subset="TRUE",
ident.1 = "WT_Stroke", ident.2 = "WT_Ctrl",
test.use = "LR", logfc.threshold = 0)
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display_tab(DEG_Strokebins_Wt_CtrlvsStroke)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
DEG_Strokebins_Appps1_CtrlvsStroke <-FindMarkers(samples.integrated.filt, subset.ident = "Stroke_bin", subset="TRUE",
ident.1 = "APPPS1_Stroke", ident.2 = "APPPS1_Ctrl",
test.use = "LR", logfc.threshold = 0)
Warning: The following arguments are not used: norm.method, subset
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display_tab(DEG_Strokebins_Appps1_CtrlvsStroke)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
genelist_both= intersect(rownames(DEG_Strokebins_Appps1_CtrlvsStroke), rownames(DEG_Strokebins_Wt_CtrlvsStroke))
plotdata=data.frame(WT=DEG_Strokebins_Wt_CtrlvsStroke[genelist_both,]$avg_log2FC, APPPS1 = DEG_Strokebins_Appps1_CtrlvsStroke[genelist_both,]$avg_log2FC)
rownames(plotdata) = genelist_both
plotdata$label = rownames(plotdata)
n=5
plotdata$label[! c(1:nrow(plotdata)) %in%
c(
order(abs(plotdata$WT), decreasing = T)[1:n],
order(abs(plotdata$APPPS1), decreasing = T)[1:n])]=""
plotdata$col="WT"
apps1=abs(plotdata$APPPS1)>abs(plotdata$WT)
plotdata$col[ apps1]="APPPS1"
plotdata$col[(abs(plotdata$APPPS1)<0.25) & (abs(plotdata$WT)<0.25)] <- NA
ggplot(plotdata, aes(x=WT, y=APPPS1, color=col, label=label))+geom_point(alpha=0.7)+
geom_text(vjust=-0.7, color="black")+geom_abline(slope=-1, intercept=0, lty=2)+geom_abline(slope=1, intercept=0, lty=2)+theme_classic()+ggtitle("log_FC_Stroke_bins")

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
to do selection of DAMS only compare APPPS1CTRL vs APPS1 Stroke to do selection of DAMs only compare APPPS1Stroke vs WT_Stroke
reads = samples.integrated.filt@assays$SCT@counts
reads.filt= reads[cluster.genes,]
datatoplot_mean = aggregate(t(reads.filt),
list(paste0(samples.integrated.filt$labels, "/",samples.integrated.filt$Genotype_Treatment)),
mean, na.rm=T) %>%
as.data.frame() %>%
column_to_rownames("Group.1") %>% scale()
datatoplot_mean=melt(datatoplot_mean)
Warning in melt(datatoplot_mean): The melt generic in data.table has been
passed a matrix and will attempt to redirect to the relevant reshape2 method;
please note that reshape2 is deprecated, and this redirection is now deprecated
as well. To continue using melt methods from reshape2 while both libraries are
attached, e.g. melt.list, you can prepend the namespace like
reshape2::melt(datatoplot_mean). In the next version, this warning will become
an error.
datatoplot_mean$Microglia = factor(gsub("/.*", "", datatoplot_mean$Var1), levels=rev(levels(samples.integrated.filt$labels)))
datatoplot_mean$Genotype_Treatment = factor(gsub(".*/", "", datatoplot_mean$Var1), levels=c("WT_Ctrl", "WT_Stroke", "APPPS1_Ctrl","APPPS1_Stroke") )
colnames(datatoplot_mean) = c("Cluster_Condition", "Gene", "Scaled_mean", "Microglia", "Genotype_Treatment")
datatoplot_perc = aggregate(t(reads.filt), list(paste0(samples.integrated.filt$labels, "/",samples.integrated.filt$Genotype_Treatment)),
function(x){length(which(x>0))/length(x)})
datatoplot_perc=melt(datatoplot_perc, value.name = "perc")
Warning in melt(datatoplot_perc, value.name = "perc"): The melt generic in
data.table has been passed a data.frame and will attempt to redirect to the
relevant reshape2 method; please note that reshape2 is deprecated, and this
redirection is now deprecated as well. To continue using melt methods from
reshape2 while both libraries are attached, e.g. melt.list, you can prepend the
namespace like reshape2::melt(datatoplot_perc). In the next version, this
warning will become an error.
Using Group.1 as id variables
datatoplot = cbind(datatoplot_mean, perc=datatoplot_perc$perc)
p <- ggplot(datatoplot, aes(x=Gene, y=Microglia, col=Scaled_mean, size=perc))+geom_point()+scale_size_continuous(range = c(0,3))+
theme_classic()+theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))+facet_grid(~Genotype_Treatment)
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/output/Microglia_Dotplot_ClsuterCondition_Genelists.svg", p)
Saving 18 x 5 in image
samples.integrated.filt.meta = samples.integrated.filt@meta.data
sce.microglia=as.SingleCellExperiment(samples.integrated.filt)
sce.microglia <- slingshot(sce.microglia, clusterLabels = 'labels', reducedDim = 'UMAP', start.clus="Microglia2")
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
Warning: useNames = NA is deprecated. Instead, specify either useNames = TRUE
or useNames = FALSE.
samples.integrated.filt.meta$Pseudotime_1 <- sce.microglia$slingPseudotime_1
samples.integrated.filt.meta$Pseudotime_2 <- sce.microglia$slingPseudotime_2
samples.integrated.filt.meta$Pseudotime_3 <- sce.microglia$slingPseudotime_3
samples.integrated.filt.meta -> samples.integrated.filt@meta.data
dataset = data.frame(reducedDim(sce.microglia, "UMAP"),
cluster = as.character(sce.microglia$labels),
DAMs = as.character(sce.microglia$DAM_binary))
curves <- slingCurves(sce.microglia, as.df=T)
p = ggplot(dataset, aes(x=umap_1, y=umap_2))+
geom_point(aes(col = cluster), size=2)+
geom_point(data=subset(dataset, dataset$DAMs==T), aes(fill=DAMs), pch=21, size=0.7)+
scale_fill_manual(values="black")
curves = curves %>% arrange(Lineage, Order)
p = p + geom_path(data = curves, aes(group = Lineage), lwd=1, lineend = "butt",)+theme_classic()
curves_arrows = curves %>% group_by(Lineage) %>% mutate(xend = lead(umap_1), yend = lead(umap_2))
curves_arrows = curves_arrows[curves_arrows$Order %in% seq(10,150, by=floor((150-10)/10)),]
p = p + geom_segment(data = curves_arrows,
aes(x = umap_1, y = umap_2, xend = xend, yend = yend),
linewidth=0.5,
arrow = arrow(length=unit(0.3, "cm"), type="closed"))
p
Warning: Removed 3 rows containing missing values (`geom_segment()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime_linages.svg", p)
Saving 7 x 5 in image
Warning: Removed 3 rows containing missing values (`geom_segment()`).
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime_linages.pdf", p)
Saving 7 x 5 in image
Warning: Removed 3 rows containing missing values (`geom_segment()`).
library(TSCAN)
n.top.gene = 10
sce.microglia.PT1<- testPseudotime(sce.microglia, pseudotime = sce.microglia$slingPseudotime_1)
sce.microglia.PT2<- testPseudotime(sce.microglia, pseudotime = sce.microglia$slingPseudotime_2)
sce.microglia.PT3<- testPseudotime(sce.microglia, pseudotime = sce.microglia$slingPseudotime_3)
By default, estimates of the spline coefficients are not returned as they are difficult to interpret. Rather, a log-fold change of expression along each path is estimated to provide some indication of the overall magnitude and direction of any change.
sce.microglia.PT1 <- sce.microglia.PT1[order(sce.microglia.PT1$FDR, decreasing = F),]
display_tab(as.data.frame(sce.microglia.PT1))
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
PT1_up = sce.microglia.PT1[sce.microglia.PT1$logFC>0,]
PT1_low = sce.microglia.PT1[sce.microglia.PT1$logFC<0,]
p <- FeaturePlot(samples.integrated.filt, features = "Pseudotime_1")+scale_color_viridis()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime1.svg", p)
Saving 7 x 5 in image
a=scater::plotExpression(sce.microglia, features=rownames(PT1_up)[1:n.top.gene], x="slingPseudotime_1", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
b=scater::plotExpression(sce.microglia, features=rownames(PT1_low)[1:n.top.gene], x="slingPseudotime_1", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
a|b
Warning: Removed 2250 rows containing missing values (`geom_point()`).
Removed 2250 rows containing missing values (`geom_point()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime1_Top10up_down_reg_Genes-correct.svg", p)
Saving 8 x 5 in image
sce.microglia.PT2 <- sce.microglia.PT2[order(sce.microglia.PT2$FDR, decreasing = F),]
display_tab(as.data.frame(sce.microglia.PT2))
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
PT2_up = sce.microglia.PT2[sce.microglia.PT2$logFC>0,]
PT2_low = sce.microglia.PT2[sce.microglia.PT2$logFC<0,]
p <- FeaturePlot(samples.integrated.filt, features = "Pseudotime_2")+scale_color_viridis()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime2.svg", p)
Saving 7 x 5 in image
a<-scater::plotExpression(sce.microglia, features=rownames(PT2_up)[1:n.top.gene], x="slingPseudotime_2", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
b<-scater::plotExpression(sce.microglia, features=rownames(PT2_low)[1:n.top.gene], x="slingPseudotime_2", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
a|b
Warning: Removed 2770 rows containing missing values (`geom_point()`).
Removed 2770 rows containing missing values (`geom_point()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime2_Top10up_down_reg_Genes.svg", a|b)
Saving 8 x 5 in image
Warning: Removed 2770 rows containing missing values (`geom_point()`).
Removed 2770 rows containing missing values (`geom_point()`).
sce.microglia.PT3 <- sce.microglia.PT3[order(sce.microglia.PT3$FDR, decreasing = F),]
display_tab(as.data.frame(sce.microglia.PT3))
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html
PT3_up = sce.microglia.PT3[sce.microglia.PT3$logFC>0,]
PT3_low = sce.microglia.PT3[sce.microglia.PT3$logFC<0,]
p <- FeaturePlot(samples.integrated.filt, features = "Pseudotime_3")+scale_color_viridis()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime3.svg", p)
Saving 7 x 5 in image
a<-scater::plotExpression(sce.microglia, features=rownames(PT3_up)[1:n.top.gene], x="slingPseudotime_3", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
b<-scater::plotExpression(sce.microglia, features=rownames(PT3_low)[1:n.top.gene], x="slingPseudotime_3", colour_by="labels")+scale_color_hue()
Scale for colour is already present.
Adding another scale for colour, which will replace the existing scale.
a|b
Warning: Removed 3420 rows containing missing values (`geom_point()`).
Removed 3420 rows containing missing values (`geom_point()`).

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(filename = "/files/scSeq_Hefendehl/docs/Pseudotime3_Top10up_down_reg_Genes.svg", a|b)
Saving 8 x 5 in image
Warning: Removed 3420 rows containing missing values (`geom_point()`).
Removed 3420 rows containing missing values (`geom_point()`).
In the paper https://www.nature.com/articles/s41586-018-0023-4 this analysis was used to generate different modules of gene lists that can then be mapped onto different treatment/genotypes within one experiment (see Figure 4). This is an analysis that can be done in Seurat https://smorabit.github.io/tutorials/9_scWGCNA_tutorial/ Which could be very cool for our data. In the paper they also have 3 different conditions in 2 genotypes.
avoid running this section as it takes very long
nclusters <- length(unique(samples.integrated.filt$labels))
genes.use <- rownames(samples.integrated.filt)
targets <- samples.integrated.filt@meta.data
group <- as.factor(samples.integrated.filt$Genotype_Treatment)
force.recalc = T
powers = c(seq(1,10,by=1), seq(12,20, by=2));
if(force.recalc | (! file.exists(file="/files/scSeq_Hefendehl/output/net.rds"))){ # skips recalculation of net object as it takes long...
datExpr <- as.data.frame(GetAssayData(samples.integrated.filt, assay='RNA', slot='data')[genes.use,])
datExpr <- as.data.frame(t(datExpr))
datExpr <- datExpr[,goodGenes(datExpr)]
# expressed in more than 10% of cells
exprgenes = colSums(datExpr>0)>nrow(datExpr)*0.1
datExpr <- datExpr[,exprgenes]
#annotated only
mmacc=as.list(org.Mm.egALIAS2EG)
annotatedgenes = colnames(datExpr) %in% names(mmacc)
datExpr <- datExpr[,annotatedgenes]
powers = c(seq(1,10,by=1), seq(12,20, by=2));
enableWGCNAThreads()
# Call the network topology analysis function for each set in turn
powerTable = list(
data = pickSoftThreshold(
datExpr,
powerVector=powers,
verbose = 100,
networkType="unsigned",
corFnc="cor"
)[[2]]
);
colors = c("blue", "red","black")
# Will plot these columns of the returned scale free analysis tables
plotCols = c(2,5,6,7)
colNames = c("Scale Free Topology Model Fit", "Mean connectivity", "mean connectivity",
"Max connectivity");
# Get the minima and maxima of the plotted points
ylim = matrix(NA, nrow = 2, ncol = 4);
for (col in 1:length(plotCols)){
ylim[1, col] = min(ylim[1, col], powerTable$data[, plotCols[col]], na.rm = TRUE);
ylim[2, col] = max(ylim[2, col], powerTable$data[, plotCols[col]], na.rm = TRUE);
}
# Plot the quantities in the chosen columns vs. the soft thresholding power
par(mfcol = c(2,2));
par(mar = c(4.2, 4.2 , 2.2, 0.5))
cex1 = 0.7;
for (col in 1:length(plotCols)){
plot(powerTable$data[,1], -sign(powerTable$data[,3])*powerTable$data[,2],
xlab="Soft Threshold (power)",ylab=colNames[col],type="n", ylim = ylim[, col],
main = colNames[col]);
addGrid();
if (col==1){
text(powerTable$data[,1], -sign(powerTable$data[,3])*powerTable$data[,2],
labels=powers,cex=cex1,col=colors[1]);
} else
text(powerTable$data[,1], powerTable$data[,plotCols[col]],
labels=powers,cex=cex1,col=colors[1]);
if (col==1){
legend("bottomright", legend = 'Metacells', col = colors, pch = 20) ;
} else
legend("topright", legend = 'Metacells', col = colors, pch = 20) ;
}
softPower=3
nSets = 1
setLabels = 'OCS'
shortLabels = setLabels
multiExpr <- list()
multiExpr[['ODC']] <- list(data=datExpr)
checkSets(multiExpr) # check data size
# construct network
net=blockwiseConsensusModules(multiExpr, blocks = NULL,
maxBlockSize = 30000, ## This should be set to a smaller size if the user has limited RAM
randomSeed = 12345,
corType = "pearson",
power = softPower,
consensusQuantile = 0.3,
networkType = "unsigned",
TOMType = "signed",
TOMDenom = "min",
scaleTOMs = TRUE, scaleQuantile = 0.8,
sampleForScaling = TRUE, sampleForScalingFactor = 1000,
useDiskCache = TRUE, chunkSize = NULL,
deepSplit = 4,
pamStage=FALSE,
detectCutHeight = 0.995, minModuleSize = 50,
mergeCutHeight = 0.2,
saveConsensusTOMs = TRUE,
consensusTOMFilePattern = "ConsensusTOM-block.%b.rda")
saveRDS(net, file="/files/scSeq_Hefendehl/output/net.rds")
saveRDS(powerTable, file="/files/scSeq_Hefendehl/output/powertable.rds")
} else {
net = readRDS(file="/files/scSeq_Hefendehl/output/net.rds")
powerTable = readRDS(file="/files/scSeq_Hefendehl/output/powertable.rds")
powers = c(seq(1,10,by=1), seq(12,20, by=2));
colors = c("blue", "red","black")
# Will plot these columns of the returned scale free analysis tables
plotCols = c(2,5,6,7)
colNames = c("Scale Free Topology Model Fit", "Mean connectivity", "mean connectivity",
"Max connectivity");
# Get the minima and maxima of the plotted points
ylim = matrix(NA, nrow = 2, ncol = 4);
for (col in 1:length(plotCols)){
ylim[1, col] = min(ylim[1, col], powerTable$data[, plotCols[col]], na.rm = TRUE);
ylim[2, col] = max(ylim[2, col], powerTable$data[, plotCols[col]], na.rm = TRUE);
}
# Plot the quantities in the chosen columns vs. the soft thresholding power
par(mfcol = c(2,2));
par(mar = c(4.2, 4.2 , 2.2, 0.5))
cex1 = 0.7;
for (col in 1:length(plotCols)){
plot(powerTable$data[,1], -sign(powerTable$data[,3])*powerTable$data[,2],
xlab="Soft Threshold (power)",ylab=colNames[col],type="n", ylim = ylim[, col],
main = colNames[col]);
addGrid();
if (col==1){
text(powerTable$data[,1], -sign(powerTable$data[,3])*powerTable$data[,2],
labels=powers,cex=cex1,col=colors[1]);
} else
text(powerTable$data[,1], powerTable$data[,plotCols[col]],
labels=powers,cex=cex1,col=colors[1]);
if (col==1){
legend("bottomright", legend = 'Metacells', col = colors, pch = 20) ;
} else
legend("topright", legend = 'Metacells', col = colors, pch = 20) ;
}
}
Warning: The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
ℹ Please use the `layer` argument instead.
This warning is displayed once every 8 hours.
Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
generated.
..Excluding 4 genes from the calculation due to too many missing samples or zero variance.
Allowing parallel execution with up to 15 working processes.
pickSoftThreshold: will use block size 5225.
pickSoftThreshold: calculating connectivity for given powers...
..working on genes 1 through 5225 of 8562
..working on genes 5226 through 8562 of 8562
Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k.
1 1 0.950 -4.140 0.970 3.49e+02 3.12e+02 1200.00
2 2 0.982 -2.710 0.994 2.73e+01 1.89e+01 265.00
3 3 0.977 -1.960 0.987 3.74e+00 1.54e+00 85.50
4 4 0.992 -1.520 0.990 8.94e-01 1.55e-01 38.10
5 5 0.988 -1.340 0.986 3.34e-01 1.80e-02 24.60
6 6 0.988 -1.250 0.985 1.67e-01 2.31e-03 18.90
7 7 0.986 -1.220 0.982 1.00e-01 3.32e-04 15.10
8 8 0.931 -1.190 0.911 6.70e-02 5.30e-05 12.40
9 9 0.943 -1.140 0.928 4.82e-02 8.90e-06 10.40
10 10 0.981 -1.080 0.975 3.65e-02 1.57e-06 8.83
11 12 0.910 -1.080 0.884 2.32e-02 5.30e-08 6.55
12 14 0.952 -1.010 0.939 1.62e-02 2.05e-09 4.99
13 16 0.954 -0.994 0.952 1.21e-02 8.53e-11 3.87
14 18 0.945 -0.962 0.962 9.50e-03 3.66e-12 3.04
15 20 0.923 -0.989 0.921 7.75e-03 1.62e-13 3.00

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
Calculating consensus modules and module eigengenes block-wise from all genes
Calculating topological overlaps block-wise from all genes
Flagging genes and samples with too many missing values...
..step 1
TOM calculation: adjacency..
..will use 15 parallel threads.
Fraction of slow calculations: 0.000000
..connectivity..
..matrix multiplication (system BLAS)..
..normalization..
..done.
..Working on block 1 .
..Working on block 1 .
..merging consensus modules that are too close..
consMEs = net$multiMEs;
moduleLabels = net$colors;
# Convert the numeric labels to color labels
moduleColors = as.character(moduleLabels)
consTree = net$dendrograms[[1]];
# module eigengenes
MEs=moduleEigengenes(multiExpr[[1]]$data, colors = moduleColors, nPC=1)$eigengenes
MEs=orderMEs(MEs)
meInfo<-data.frame(rownames(datExpr), MEs)
colnames(meInfo)[1]= "SampleID"
# intramodular connectivity
KMEs<-signedKME(datExpr, MEs,outputColumnName = "kME",corFnc = "bicor")
Warning in bicor(datExpr, datME, , use = "p"): bicor: zero MAD in variable 'x'.
Pearson correlation was used for individual columns with zero (or missing) MAD.
# compile into a module metadata table
geneInfo=as.data.frame(cbind(colnames(datExpr),moduleColors, KMEs))
# how many modules did we get?
nmodules <- length(unique(moduleColors))
# merged gene symbol column
colnames(geneInfo)[1]= "GeneSymbol"
colnames(geneInfo)[2]= "Initially.Assigned.Module.Color"
# save info
write.csv(geneInfo,file=paste0('/files/scSeq_Hefendehl/output/geneInfoSigned.csv'))
PCvalues=MEs
p = WGCNA::plotDendroAndColors(consTree, moduleColors[net$goodGenes], "Module colors", dendroLabels = FALSE, hang = 0.03, addGuide = TRUE, guideHang = 0.05,
main = paste0("Microglia gene dendrogram and module colors"))

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(file="/files/scSeq_Hefendehl/output/Microglia_WGCNA_Dendrocolplot.svg")
Saving 7 x 5 in image
Warning: Removed 3420 rows containing missing values (`geom_point()`).
Removed 3420 rows containing missing values (`geom_point()`).
## Todo adjust
plot_df <- cbind(dplyr::select(targets, c(Genotype, Treatment)), PCvalues)
plot_df <- reshape2::melt(plot_df, id.vars = c('Genotype', 'Treatment'))
plot_df$Genotype <- factor(plot_df$Genotype, levels=c('WT','APPPS1'))
plot_df$Genotype_Treatment <- paste0(plot_df$Genotype, "_", plot_df$Treatment)
plot_df$Genotype_Treatment <- factor(plot_df$Genotype_Treatment, levels=c('WT_Ctrl','WT_Stroke', "APPPS1_Ctrl", "APPPS1_Stroke"))
colors <- sub('ME', '', as.character(levels(plot_df$variable)))
p <- ggplot(plot_df, aes(x=variable, y=value, fill=Treatment)) +
geom_boxplot(notch=FALSE) +
RotatedAxis() + ylab('Module Eigengene') + xlab('') +
theme(
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),
)
p <- p + facet_wrap(~variable+Genotype, scales='free_x', ncol=nmodules)+theme_classic()
p

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
p2 <- ggplot(plot_df, aes(x=variable, y=value, fill=Genotype_Treatment)) +
geom_violin() +
# RotatedAxis() +
ylab('Module Eigengene') + xlab('') +
theme(
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),
)
p2 <- p2 + facet_wrap(~variable, scales='free', ncol=nmodules)+theme_classic()
p2

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
ggsave(file="/files/scSeq_Hefendehl/output/Microgila_WGCNA_Eigenscore.svg", p2)
Saving 6 x 2 in image
reslist = list()
for(M in unique(plot_df$variable)){
Mdata = plot_df[plot_df$variable==M,]
fit= lm(value~Genotype*Treatment, data = Mdata)
resfit = summary(fit)
reslist[[M]] <- c(Genotype_Treatment = resfit$coefficients["GenotypeAPPPS1:TreatmentStroke", "Pr(>|t|)"])
}
ME_avg <- plot_df %>% dplyr::group_by(variable, Treatment, Genotype) %>% summarise(avg_Eigenmodule = mean(value),
sd_Eigenvalue = sd(value),
med_Eigenvalue=median(value))
`summarise()` has grouped output by 'variable', 'Treatment'. You can override
using the `.groups` argument.
ME_avg$pval=unlist(reslist[ME_avg$variable])
ME_avg$pval_ast=convertpvaltostars(ME_avg$pval)
ME_avg$Genotype_Treatment = paste0(ME_avg$Genotype, "_",ME_avg$Treatment)
ME_avg$Genotype_Treatment <- factor(ME_avg$Genotype_Treatment, levels=c('WT_Ctrl','WT_Stroke', "APPPS1_Ctrl", "APPPS1_Stroke"))
ME_avg$Module = paste0(ME_avg$pval_ast, ME_avg$variable)
ggplot(ME_avg, aes(x=Genotype_Treatment, y=Module, fill=avg_Eigenmodule))+geom_tile()+scale_fill_viridis()+theme_classic()+theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))

| Version | Author | Date |
|---|---|---|
| 23643ee | achiocch | 2024-02-09 |
Meandata = MEs %>% group_by(samples.integrated.filt$Genotype_Treatment) %>% summarise_all(mean) %>% column_to_rownames("samples.integrated.filt$Genotype_Treatment") %>% t()
SDdata = MEs %>% group_by(samples.integrated.filt$Genotype_Treatment) %>% summarise_all(sd)%>% column_to_rownames("samples.integrated.filt$Genotype_Treatment") %>% t()
colLabels=colnames(Meandata)
rowLabels=rownames(Meandata)
textlab = paste0(signif(Meandata,2), "\n", "(", signif(SDdata, 2), ")")
svg("/files/scSeq_Hefendehl/output/WGCNA_labeled_Heatmap.svg")
labeledHeatmap(Meandata, xLabels = colLabels,
yLabels = rowLabels,
colorLabels = TRUE,
colors = viridis(50),
setStdMargins = FALSE, textMatrix = textlab,
cex.col="white",
main = "Mean(SD) Eigengene-expression");
Warning in plot.window(xlim, ylim, log = log, ...): "cex.col" is not a
graphical parameter
Warning in title(main = main, sub = sub, xlab = xlab, ylab = ylab, ...):
"cex.col" is not a graphical parameter
dev.off()
png
2
Modules = unique(moduleColors)
Modules = Modules[! Modules %in% "grey"] # drop grey one
for(m in Modules){
p = DoHeatmap(samples.integrated.filt, features = rownames(samples.integrated.filt)[moduleColors==m], slot="counts",
group.by = "Genotype_Treatment")+scale_fill_viridis()
ggsave(paste0("/files/scSeq_Hefendehl/output/WGCNAGeneWise_ExpressionModule", m, ".svg"), p)
}
Scale for fill is already present.
Adding another scale for fill, which will replace the existing scale.
Saving 7 x 5 in image
Scale for fill is already present.
Adding another scale for fill, which will replace the existing scale.
Saving 7 x 5 in image
Scale for fill is already present.
Adding another scale for fill, which will replace the existing scale.
Saving 7 x 5 in image
Scale for fill is already present.
Adding another scale for fill, which will replace the existing scale.
Saving 7 x 5 in image
Scale for fill is already present.
Adding another scale for fill, which will replace the existing scale.
Saving 7 x 5 in image
genelist = split(names(net$colors), net$colors)
gene_univers = names(net$colors)
pvallist = unlist(reslist)
MOI = gsub("ME", "", names(pvallist)) %>% gsub(".Genotype_Treatment", "", .)
# MOI = MOI[pvallist<0.05]
# if you want to have the affected genes set return_genelist = T; this takes roughly 4-8 hours
Gores = getGOresults(geneset = genelist[MOI], domain_scope = "known",
genereference = gene_univers,
return_genelist = T,
organism = "mmusculus")
Detected custom background input, domain scope is set to 'custom'
display_tab(Gores$result)
Warning in instance$preRenderHook(instance): It seems your data is too big for
client-side DataTables. You may consider server-side processing:
https://rstudio.github.io/DT/server.html